Complexation of nucleic acids with disulfide-crosslinked cationic components for transfection and immunostimulation

ABSTRACT

The present invention is directed to a polymeric carrier cargo complex, comprising as a cargo at least one nucleic acid (molecule) and disulfide-crosslinked cationic components as a (preferably non-toxic and non-immunogenic) polymeric carrier. The inventive polymeric carrier cargo complex allows for both efficient transfection of nucleic acids into cells in vivo and in vitro and/or for induction of an (innate and/or adaptive) immune response, preferably dependent on the nucleic acid to be transported as a cargo. The present invention also provides, pharmaceutical compositions, particularly vaccines and adjuvants, comprising the inventive polymeric carrier cargo complex and optionally an antigen, as well as the use of such the inventive polymeric carrier cargo complex and optionally an antigen for transfecting a cell, a tissue or an organism, for (gene-)therapeutic purposes as disclosed herein, and/or as an immunostimulating agent or adjuvant, e.g. for eliciting an immune response for the treatment or prophylaxis of diseases as mentioned above. Finally, the invention relates to kits containing the inventive polymeric carrier cargo complex and/or the inventive pharmaceutical composition, adjuvant or vaccine in one or more parts of the kit.

This application is a continuation of U.S. application Ser. No.14/600,628, filed Jan. 20, 2015, which is a continuation of U.S.application Ser. No. 13/203,653, filed Feb. 14, 2013, now U.S. Pat. No.8,968,746, which is a national phase application under 35 U.S.C. § 371of International Application No. PCT/EP2011/003719, filed Jul. 25, 2011,which claims benefit of and priority to U.S. Provisional Application No.61/369,549, filed Jul. 30, 2010, and European Application No.10007992.0, filed Jul. 30, 2010. The entire text of each of the abovereferenced disclosures is specifically incorporated herein by reference.

This application contains a Sequence Listing XML, which has beensubmitted electronically and is hereby incorporated by reference in itsentirety. Said Sequence Listing XML, created on Nov. 3, 2022, is namedCRVCP0094USC2.xml and is 650,106 bytes in size.

The present invention is directed to a polymeric carrier cargo complex,comprising as a cargo at least one nucleic acid (molecule) and as a(preferably non-toxic and non-immunogenic) polymeric carrierdisulfide-crosslinked cationic components. The inventive polymericcarrier cargo complex allows for both efficient transfection of nucleicacids into cells in vivo and in vitro and/or for induction of an (innateand/or adaptive) immune response, preferably dependent on the nucleicacid to be transported as a cargo. The present invention also providespharmaceutical compositions, particularly vaccines, comprising theinventive polymeric carrier cargo complex and optionally an antigen, aswell as the use of the inventive polymeric carrier cargo complex andoptionally an antigen for transfecting a cell, a tissue or an organism,for (gene-) therapeutic purposes as disclosed herein, and/or as animmunostimulating agent or adjuvant, e.g. for eliciting an immuneresponse for the treatment or prophylaxis of diseases as mentionedabove. Finally, the invention relates to kits containing the inventivepolymeric carrier cargo complex, the inventive pharmaceuticalcomposition and/or the inventive vaccine or any of its components in oneor more parts of the kit.

Many diseases today require administration of adjuvants to provide aninnate immune response and, optionally, to support an adaptive immuneresponse, particularly in the context of vaccinations. Some but notnecessarily all of these diseases additionally or alternatively requireadministration of peptide-, protein-, and nucleic acid-based drugs, e.g.the transfection of nucleic acids into cells or tissues. Theserequirements usually represent different aspects in the treatment ofsuch diseases and are typically difficult to address in one approach. Asa consequence, the prior art usually handles such aspects via separateapproaches.

In the above context, vaccination is generally believed to be one of themost effective and cost-efficient ways to prevent or treat diseases.Nevertheless several problems in vaccine development have proveddifficult to solve: Vaccines are often inefficient for the very youngand the very old; many vaccines need to be given several times, and theprotection they confer wanes over time, requiring boosteradministrations, and, for some diseases such as HIV, development ofefficient vaccines is urgently needed. As generally accepted, many ofthese vaccines would be enabled or improved if they could elicit astronger and more durable immune response.

Accordingly, the development of new efficient and safe adjuvants forvaccination purposes which support induction and maintenance of anadaptive immune response by initiating or boosting a parallel innateimmune response represents a main challenging problem.

Adjuvants are usually defined as compounds that can increase and/ormodulate the intrinsic immunogenicity of an antigen. To reduce negativeside effects, new vaccines have a more defined composition that oftenleads to lower immunogenicity compared with previous whole-cell orvirus-based vaccines. Adjuvants are therefore required to assist newvaccines to induce potent and persistent immune responses, with theadditional benefit that less antigen and fewer injections are needed.Now it is clear that the adaptive immune response mainly depends on thelevel and specificity of the initial danger signals perceived by innateimmune cells following infection or vaccination (Guy, B. (2007), Nat RevMicrobiol 5(7): 505-17). In particular for new generation vaccinecandidates, which will increasingly comprise highly purified recombinantproteins and, although very safe, are poorly immunogenic, efficientadjuvants will become increasingly necessary.

Unfortunately, only a few licensed adjuvants are available so far. Mostprominent is Alum, which is known to be safe, but also represents a veryweak adjuvant. Many further adjuvants have been developed, e.g.including the administration of pathogens, CpG-nucleotides, etc. Most ofthese new or “established” adjuvants, however, still do not satisfy theabove requirements, since many new and emerging problems have to beconsidered and solved. These problems inter alia include new andre-emerging infectious diseases, repeated administrations, threat ofpandemic flu, etc.

Furthermore, the new vaccine targets are usually more difficult todevelop and—due to their specifically tailored immune responses—requiremore potent adjuvants to enable success. Moreover, there are still asignificant number of important pathogens for which we do not even haveeffective vaccines at present. This represents a very challenging futuretarget. To enable vaccine development against such targets, more potentadjuvants will be necessary. Such new adjuvants will need to offeradvantages, including more heterologous antibody responses, coveringpathogen diversity, induction of potent functional antibody responses,ensuring pathogen killing or neutralization and induction of moreeffective T cell responses, for direct and indirect pathogen killing,particularly the induction of cytotoxic T cells which are part of a Th1immune response. In addition, adjuvants may be necessary to achieve morepragmatic effects, including antigen dose reduction and overcomingantigen competition in combination vaccines. Moreover, against thebackground of an aging population, which is increasingly susceptible toinfectious diseases, new adjuvants will be necessary to overcome thenatural deterioration of the immune response with age (O'Hagan, D. T.and E. De Gregorio (2009), Drug Discov Today 14(11-12): 541-51).

The review of O'Hagan (2009; supra) summarizes some reasons for theurgent need of new effective adjuvants e.g. the requirement of a lowerantigen dose in vaccines, the necessity to increase the breadth of animmune response and the heterologous activity, to enable complexcombination vaccines, and to overcome antigenic competition, to overcomelimited immune response in some groups of the population, such as theelderly, the young children, and infants, patients with chronic diseasesand the immunocompromised, to increase effector T cell response andantibody titers, to induce protective responses more rapidly and also toextend the duration of response by enhancing memory B and T cellresponses.

Summarizing the above, new efficient and safe immunostimulating agentsor adjuvants are required, which are preferably efficient in inducing aninnate immune response, particularly in inducing the anti-viral cytokineIFN-alpha; which are, preferably, also efficient in supporting anadaptive immune response; safe, i.e. not associated with any long-termeffects; which are well tolerated; which are available via a simplesynthetic pathway; which exhibit low cost storage conditions(particularly feasible lyophilisation); which require simple andinexpensive components; which are biodegradable; which are compatiblewith many different kinds of vaccine antigens; which are capable ofcodelivery of antigen and immune potentiator, etc.

As already explained above adjuvants or immunostimulating agents usuallyact via their capability to induce an innate immune response. The innateimmune system forms the dominant system of host defense in mostorganisms and comprises barriers such as humoral and chemical barriersincluding, e.g., inflammation, the complement system and cellularbarriers. The innate immune system is typically based on a small numberof receptors, called pattern recognition receptors. They recognizeconserved molecular patterns that distinguish foreign organisms, likeviruses, bacteria, fungi and parasites, from cells of the host. Suchpathogen-associated molecular patterns (PAMP) include viral nucleicacids, components of bacterial and fungal walls, flagellar proteins, andmore. The first family of pattern recognition receptors (PAMP receptors)studied in detail was the Toll-like receptor (TLR) family. TLRs aretransmembrane proteins which recognize ligands of the extracellularmilieu or of the lumen of endosomes. Following ligand-binding theytransduce the signal via cytoplasmic adaptor proteins which leads totriggering of a host-defence response and entailing production ofantimicrobial peptides, proinflammatory chemokines and cytokines,antiviral cytokines, etc. (see e.g. Meylan, E., J. Tschopp, et al.(2006), Nature 442(7098): 39-44). Further relevant components of theimmune system include e.g. the endosomal TLRs, cytoplasmic receptors,Type I interferons and cytoplasmic receptors. Therefore, theimmunostimulating agents or adjuvants are defined herein preferably asinducers of an innate immune response, which active pattern recognitionreceptors (PAMP receptors). Hereby, a cascade of signals is elicited,which e.g. may result in the release of cytokines (e.g. IFN-alpha)supporting the innate immune response. Accordingly, it is preferably afeature of an immunostimulating agent or adjuvant to bind to suchreceptors and activate such PAMP receptors. Ideally, such as an agent oradjuvant additionally supports the adaptive immune response by e.g.shifting the immune response such that the preferred class of Th cellsis activated. Depending on the disease or disorder to be treated a shiftto a Th1-based immune response may be preferred or, in other cases, ashift to a Th2 immune response may be preferred.

In the prior art there are some promising adjuvant candidates whichfulfil at least some, but not all, of the above defined requiredcharacteristics.

As an example, among the above developed new adjuvants, some nucleicacids like CpG DNA oligonucleotides or isRNA (immunostimulating RNA)turned out to be promising candidates for new immunostimulating agentsor adjuvants as they allow the therapeutic or prophylactic induction ofan innate immune response. Comprehensibly, such nucleic acid basedadjuvants usually have to be delivered effectively to the site of actionto allow induction of an effective innate immune response withoutunnecessary loss of adjuvant activity and, in some cases, without thenecessity to increase the administered volume above systemicallytolerated levels.

One approach to solve this issue may be the transfection of cells whichare part of the innate immune system (e.g. dendritic cells, plasmacytoiddendritic cells (pDCs)) with immunostimulatory nucleic acids, which areligands of PAMP receptors, (e.g. Toll-like receptors (TLRs)), and thusmay lead to immunostimulation by the nucleic acid ligand. Furtherapproaches may be the direct transfection of nucleic acid basedadjuvants. All of these approaches, however, are typically impaired byinefficient delivery of the nucleic acid and consequently diminishedadjuvant activity, in particular when administered locally.

However, one main disadvantage of such nucleic acid based adjuvantapproaches until today is their limited ability to cross the plasmamembrane of mammalian cells, resulting in poor cellular access andinadequate therapeutic efficacy. Until today this hurdle represents amajor challenge for nucleic acid transfection based applications, e.g.biomedical developments and accordingly the commercial success of manybiopharmaceuticals (see e.g. Foerg, C. & Merkle, H. P., J Pharm Sci 97,144-62 (2008).

Transfection of nucleic acids or genes into cells or tissues has beeninvestigated up to date in the context of in vitro transfection purposesand in the context of gene therapeutic approaches. However, no adjuvantsare available so far which are based on such gene delivery techniqueswhich are efficient and safe, in particular no licensed adjuvants. Thisis presumably due to the complex requirements of adjuvants in general incombination with stability issues to be solved in the case of nucleicacid based adjuvants.

Nevertheless, transfection of nucleic acids or genes into cells ortissues for eliciting an (innate and/or adaptive) immune responseappears to provide a promising approach to provide new adjuvants.

However, many of these approaches utilize transfection of nucleic acidsor genes into cells or tissues without induction of an innate immuneresponse. There even some gene therapeutic therapies, which have tostrictly avoid induction of an innate immune response. Even in the rarecases, where vaccination is carried out to induce an adaptiveantigen-specific immune response using administration of nucleic acids,e.g. in tumour vaccinations using DNA or mRNA encoded antigens,induction of an adaptive immune response is typically carried out as anactive immunization against the encoded antigen but not as anaccompanying adjuvant therapy and thus requires additionaladministration of a separate adjuvant to induce an innate immuneresponse.

Even if a lot of transfection methods are known in the art, transfer orinsertion of nucleic acids or genes into an individual's cells stillrepresents a major challenge today and is not yet solved satisfactorily.To address this complex issue a variety of methods were developed in thelast decade. These include transfection by calcium phosphate, cationiclipids, cationic polymers, and liposomes. Further methods fortransfection are electroporation and viral transduction.

However, as known to a skilled person, systems for transfer or insertionof nucleic acids or genes have to fulfil several requirements for invivo applications which include efficient nucleic acid delivery into anindividual's cells with high functionality, protection of the nucleicacid against ubiquitously occurring nucleases, release of the nucleicacid in the cell, no safety concerns, feasible manufacturing in acommercially acceptable form amenable to scale-up and storage stabilityunder low cost conditions (e.g feasible lyophilisation). Theserequirements are to be added to the complex requirements of an adjuvantparticularly if it is in the form of a nucleic acid as outlined above.

Some successful strategies for the transfer or insertion of nucleicacids or genes available today rely on the use of viral vectors, such asadenoviruses, adeno-associated viruses, retroviruses, and herpesviruses. Viral vectors are able to mediate gene transfer with highefficiency and the possibility of long-term gene expression. However,the acute immune response (“cytokine storm”), immunogenicity, andinsertion mutagenesis uncovered in gene therapy clinical trials haveraised serious safety concerns about some commonly used viral vectors.

Another solution to the problem of transfer or insertion of nucleicacids or genes may be found in the use of non-viral vectors. Althoughnon-viral vectors are not as efficient as viral vectors, many non-viralvectors have been developed to provide a safer alternative. Methods ofnon-viral nucleic acid delivery have been explored using physical(carrier-free nucleic acid delivery) and chemical approaches (syntheticvector-based nucleic acid delivery). Physical approaches usually includeneedle injection, electroporation, gene gun, ultrasound, andhydrodynamic delivery, employ a physical force that permeates the cellmembrane and facilitates intracellular gene transfer. The chemicalapproaches typically use synthetic or naturally occurring compounds(e.g. cationic lipids, cationic polymers, lipid-polymer hybrid systems)as carriers to deliver the nucleic acid into the cells. Althoughsignificant progress has been made in the basic science and applicationsof various nonviral nucleic acid delivery systems, the majority ofnon-viral approaches are still much less efficient than viral vectors,especially for in vivo gene delivery (see e.g. Gao, X_(m) Kim, K. & Liu,D., AAPS J 9, E92-104 (2007)).

Such transfection agents as defined above typically have been usedsuccessfully solely in in vitro reactions. For application of nucleicacids in vivo, however, further requirements have to be fulfilled. Forexample, complexes between nucleic acids and transfection agents have tobe stable in physiological salt solutions with respect toagglomerisation. Furthermore, such complexes typically must not interactwith parts of the complement system of the host and thus must not beimmunogenic itself as the carrier itself shall not induce an adaptiveimmune response in the individual. Additionally, the complex shallprotect the nucleic acid from early extracellular degradation byubiquitously occurring nucleases.

In the art many transfection reagents are available, especially cationiclipids, which show excellent transfection activity in cell culture.However, most of these transfection reagents do not perform well in thepresence of serum, and only a few are active in vivo. A dramatic changein size, surface charge, and lipid composition occurs when lipoplexesare exposed to the overwhelming amount of negatively charged and oftenamphipathic proteins and polysaccharides that are present in blood,mucus, epithelial lining fluid, or tissue matrix. Once administered invivo, lipoplexes tend to interact with negatively charged bloodcomponents and form large aggregates that could be absorbed onto thesurface of circulating red blood cells, trapped in a thick mucus layer,or embolized in microvasculatures, preventing them from reaching theintended target cells in the distal location. Some even undergodissolution after they are introduced to the blood circulation (see e.g.Gao, X_(m) Kim, K. & Liu, D., AAPS J 9, E92-104 (2007)).

One more promising approach utilizes cationic polymers. Cationicpolymers turned out to be efficient in transfection of nucleic acids, asthey can tightly complex and condense a negatively charged nucleic acid.Thus, a number of cationic polymers have been explored as carriers forin vitro and in vivo gene delivery. These include polyethylenimine(PEI), polyamidoamine and polypropylamine dendrimers, polyallylamine,cationic dextran, chitosan, cationic proteins and cationic peptides.Although most cationic polymers share the function of condensing DNAinto small particles and facilitate cellular uptake via endocytosisthrough charge-charge interaction with anionic sites on cell surfaces,their transfection activity and toxicity differs dramatically.

Only in one approach in the art, the immunostimulatory effect of RNAcomplexed to short cationic peptides was demonstrated by Fotin-Mleczeket al. (WO 2009/030481). These formulations appear to efficiently inducethe cytokine production in immunocompetent cells. UnfortunatelyFotin-Mleczek et al. did not assess the induction of the preferableanti-viral cytokine IFN-α by these complexes. Additionally, thesecomplexes turned out to be unstable during lyophilisation.

In the above context, cationic polymers exhibit better transfectionefficiency with rising molecular weight. However, a rising molecularweight also leads to a rising toxicity of the cationic polymer. In thisabove context, (high molecular weight) PEI is perhaps the most activeand most studied polymer for transfection of nucleic acids, inparticular for gene delivery purposes. Unfortunately, it exhibits thesame drawback due to its non-biodegradable nature and toxicity.Furthermore, even though polyplexes formed by high molecular weightpolymers exhibit improved stability under physiological conditions, datahave indicated that such polymers can hinder vector unpacking. Toovercome this negative impact, Read et al. (see Read, M. L. et al., JGene Med. 5, 232-245 (2003); and Read, M. L. et al., Nucleic Acids Res33, e86 (2005)) developed a new type of synthetic vector based on alinear reducible polycation (RPC) prepared by oxidative polycondensationof the peptide Cys-Lys₁₀-Cys (SEQ ID NO: 391). This peptideCys-Lys₁₀-Cys (SEQ ID NO: 391) can be cleaved by the intracellularenvironment to facilitate release of nucleic acids. In this context,Read et al. (2003, supra) could show that polyplexes formed by theseRPCs are destabilised by reducing conditions enabling efficient releaseof DNA and mRNA. However, examining the transfection efficiency in vitroRead et al. (2003, supra) also observed that N/P (nitrogen to phosphoratoms) ratios of 2 were unsatisfying and higher N/P ratios werenecessary to improve transfection efficiency. Additionally, Read et al.(2003, supra) observed that chloroquine or the cationic lipid DOTAP wasadditionally necessary to enhance transfection efficiency to adequatelevels. As a consequence, Read et al. (2005, supra) included histidineresidues into the RPCs which have a known endosomal buffering capacityand showed that such histidine-rich RPCs can be cleaved by theintracellular reducing environment. This approach enabled efficientcytoplasmic delivery of a broad range of nucleic acids, includingplasmid DNA, mRNA and siRNA molecules without the requirement for theendosomolytic agent chloroquine.

Unfortunately, neither Read et al. (2003, supra) nor Read et al. (2005,supra) did assess as to whether RPCs can be directly used for in vivoapplications. In their study in 2005, transfections were performed inthe absence of serum to avoid masking the ability of histidine residuesto enhance gene transfer that may have arisen from binding of serumproteins to polyplexes restricting cellular uptake. Preliminaryexperiments, however, indicated that the transfection properties ofhistidine-rich RPC polyplexes can be affected by the presence of serumproteins with a 50% decrease in GFP-positive cells observed in 10% FCS.For in vivo application Read et al. (2005, supra) proposed modificationswith the hydrophilic polymer poly-[N-(2hydroxy-propyl)methacrylamide].Unfortunately, they could not prevent aggregation of polyplexes andbinding of polycationic complexes to serum proteins. Furthermore, strongcationic charged complexes are formed (positive zeta potential) whencomplexing the nucleic acid due to the large excess of cationic polymer,which is characterized by the high N/P ratio. Accordingly, suchcomplexes are only of limited use in vivo due to their strong tendencyof salt induced agglomeration and interactions with serum contents(opsonization). Additionally, these (positively charged) complexes mayexcite complement activation, when used for purposes of gene therapy. Ithas also turned out that these positively charged RPC based complexesshowed poor translation of the nucleic acid cargo subsequent to localadministration into the dermis.

In an approach similar to Read et al. McKenzie et al. (McKenzie, D. L.,K. Y. Kwok, et al. (2000), J Biol Chem 275(14): 9970-7. and McKenzie, D.L., E. Smiley, et al. (2000), Bioconjug Chem 11(6): 901-9) developedcross-linking peptides as gene delivery agents by inserting multiplecysteines into short synthetic peptides. In their studies they examinedthe optimal complex formation with DNA and as a result they could showthat an N/P ratio of at least 2 is necessary for fully formed peptideDNA condensates. Therefore only positively charged complexes appeared toshow optimal DNA condensation. In contrast to these data they proposedthe development of negatively charged complexes for in vivo genedelivery, since it was shown in previous studies that intravenousapplication of electropositive DNA condensates leads to rapidopsonisation and nonspecific biodistribution to lung and liver (Collard,W. T., Evers, D. L., McKenzie, D. L., and Rice, K. G. (2000), Carbohydr.Res. 323, 176-184). Therefore McKenzie et al. (2000; supra) proposed thederivatization of the carriers with polyethylene glycol and targetingligands. To be noted, the approach of McKenzie et al. (2000, supra) isadditionally subject of a patent (U.S. Pat. No. 6,770,740 B1), whichparticularly discloses the transfection of coding nucleic acids,antisense nucleic acids and ribozymes.

Thus, in vivo application of nucleic acids appears to be still one ofthe most challenging problems because plasma proteins with anioniccharges may non-specifically bind to positively charged complexes andrapidly remove them e.g. via the reticulo-endothelial system.Opsonization and activation of the complement system by cationiccomplexes are additional physiological phenomena that can participate inlowering the efficacy of in vivo administered cationic complexes. Thisparticularly applies to administration of nucleic acid-based drugs, e.g.the transfection of nucleic acids into cells or tissues, particularly ifthe expression of an encoded protein or peptide or transcription of anRNA of the transfected nucleic acid is intended.

Summarizing the above, the prior art does not provide feasible means ormethods, which, on the one hand side, allow to establish efficient andsafe adjuvants for vaccination purposes, and which, on the other handside, are furthermore suited for in vivo delivery of nucleic acids, inparticular for compacting and stabilizing a nucleic acid for thepurposes of nucleic acid transfection in vivo without exhibiting thenegative side effects as discussed above. More precisely, no means ormethods are known in the prior art in the above context, which are, onthe one hand side, stable enough to carry a nucleic acid cargo to thetarget before they are metabolically cleaved, and which, on the otherhand side, can be cleared from the tissue before they can accumulate andreach toxic levels. In addition no means or method is known, which,additional to the above requirements, induces a desirable pattern ofcytokines, particularly the anti viral cytokine IFN-α.

Accordingly, it is the object of the present invention to provide suchmeans or methods, which address these problems.

The object underlying the present invention is solved by the subjectmatter of the present invention, preferably by the subject matter of theattached claims.

According to a first embodiment, the object underlying the presentinvention is solved by a polymeric carrier cargo complex, comprising

-   -   a) (as a carrier) a polymeric carrier formed by        disulfide-crosslinked cationic components, and    -   b) (as a cargo) at least one nucleic acid (molecule), preferably        for use as a medicament, more preferably for use as an        immunostimulating agent or adjuvant, e.g. in the treatment of a        disease as defined herein.

Alternatively, the object underlying the present invention is solved bya polymeric carrier cargo complex, consisting of

-   -   a) a polymeric carrier formed by disulfide-crosslinked cationic        components (as a carrier), and    -   b) at least one nucleic acid (molecule) (as a cargo), preferably        for use as a medicament, more preferably for use as an        immunostimulating agent or adjuvant, e.g. in the treatment of a        disease as defined herein.

The term “immunostimulating agent” is typically understood not toinclude agents as e.g. antigens (of whatever chemical structure), whichelicit an adaptive/cytotoxic immune response, e.g. a “humoral” or“cellular” immune response, in other words elicit immune responses (andconfer immunity by themselves) which are characterized by a specificresponse to structural properties of an antigen recognized to be foreignby immune competent cells. Rather, by “immunostimulating agent”, it istypically understood to mean agents/compounds/complexes which do nottrigger any adaptive/cytotoxic immune response by themselves, but whichmay exclusively enhance such an adaptive/cytotoxic immune response in anunspecific way, by e.g. activating “PAMP” receptors and therebytriggering the release of cytokines which support the actualadaptive/cytotoxic immune response. Accordingly, any immunostimulationby agents (e.g. antigens) which evoke an adaptive and/or cytotoxicimmune response by themselves (conferring immunity by themselvesdirectly or indirectly) is typically disclaimed by the phrase “immunostimulating agent”.

The term “adjuvant” is also understood not to comprise agents whichconfer immunity by themselves. Accordingly, adjuvants do not bythemselves confer immunity, but assist the immune system in various waysto enhance the antigen-specific immune response by e.g. promotingpresentation of an antigen to the immune system. Hereby, an adjuvant maypreferably e.g. modulate the antigen-specific immune response by e.g.shifting the dominating Th1-based antigen specific response to a moreTh2-based antigen specific response or vice versa. Accordingly, theterms “immunostimulating agent” and “adjuvant” in the context of thepresent invention are typically understood to mean agents, compounds orcomplexes which do not confer immunity by themselves, but exclusivelysupport the immune response in an unspecific way (in contrast to anantigen-specific immune response) by effects, which modulate theantigen-specific (adaptive cellular and/or humoral immune response) byunspecific measures, e.g. cytokine expression/secretion, improvedantigen presentation, shifting the nature of the arms of the immuneresponse etc. Accordingly, any agents evoking by themselves immunity aretypically disclaimed by the terms “adjuvant” or “immunostimulatingagent”.

The inventive polymeric carrier cargo complex allows provision ofefficient and safe adjuvants for vaccination purposes and carriers fortransfection, either in the field of vaccination, adjuvant therapy orgene therapeutic applications, etc. Advantageously, the inventivepolymeric carrier cargo complex is suited for in vivo delivery ofnucleic acids, in particular for compacting and stabilizing a nucleicacid for the purposes of nucleic acid transfection without exhibitingthe negative side effects of high-molecular weight polymers as discussedabove, such as no or bad biodegradability or even high toxicity,agglomeration, low transfection activity in vivo, etc. The inventivepolymeric carrier cargo complex also provides for efficient nucleic acidtransfer in vivo particularly via intradermal or intramuscular routes,including serum stability, salt stability, efficient uptake, nocomplement activation, nucleic acid release, etc. Such an inventivepolymeric carrier cargo complex, when provided as an adjuvant,furthermore supports induction and maintenance of an adaptive immuneresponse by initiating or boosting a parallel innate immune response.Additionally, the inventive polymeric carrier cargo complex exhibitsexcellent storage stability, particularly during lyophilization.

The inventive polymeric carrier cargo complex as defined above comprisesas one component a polymeric carrier formed by disulfide-crosslinkedcationic components. The term “cationic component” typically refers to acharged molecule, which is positively charged (cation) at a pH value ofabout 1 to 9, preferably of a pH value of or below 9, of or below 8, ofor below 7, most preferably at physiological pH values, e.g. about 7.3to 7.4. Accordingly, a cationic peptide, protein or polymer according tothe present invention is positively charged under physiologicalconditions, particularly under physiological salt conditions of the cellin vivo. The definition “cationic” may also refer to “polycationic”components.

In this context the cationic components, which form basis for thepolymeric carrier of the inventive polymeric carrier cargo complex bydisulfide-crosslinkage, are typically selected from any suitablecationic or polycationic peptide, protein or polymer suitable for thispurpose, particular any cationic or polycationic peptide, protein orpolymer capable to complex a nucleic acid as defined according to thepresent invention, and thereby preferably condensing the nucleic acid.The cationic or polycationic peptide, protein or polymer, is preferablya linear molecule, however, branched cationic or polycationic peptides,proteins or polymers may also be used.

Each cationic or polycationic protein, peptide or polymer of thepolymeric carrier contains at least one —SH moiety, most preferably atleast one cysteine residue or any further chemical group exhibiting an—SH moiety, capable to form a disulfide linkage upon condensation withat least one further cationic or polycationic protein, peptide orpolymer as cationic component of the polymeric carrier as mentionedherein.

Each cationic or polycationic protein, peptide or polymer or any furthercomponent of the polymeric carrier is preferably linked to itsneighbouring component(s) (cationic proteins, peptides, polymers orother components) via disulfide-crosslinking. Preferably, thedisulfide-crosslinking is a (reversible) disulfide bond (—S—S—) betweenat least one cationic or polycationic protein, peptide or polymer and atleast one further cationic or polycationic protein, peptide or polymeror other component of the polymeric carrier. The disulfide-crosslinkingis typically formed by condensation of —SH-moieties of the components ofthe polymeric carrier particularly of the cationic components. Such an—SH-moiety may be part of the structure of the cationic or polycationicprotein, peptide or polymer or any further component of the polymericcarrier prior to disulfide-crosslinking or may be added prior todisulfide-crosslinking by a modification as defined below. In thiscontext, the sulphurs adjacent to one component of the polymericcarrier, necessary for providing a disulfide bond, may be provided bythe component itself, e.g. by a —SH moiety as defined herein or may beprovided by modifying the component accordingly to exhibit a —SH moiety.These —SH-moieties are typically provided by each of the component, e.g.via a cysteine or any further (modified) amino acid or compound of thecomponent, which carries a—SH moiety. In the case that the cationiccomponent or any further component of the polymeric carrier is a peptideor protein it is preferred that the —SH moiety is provided by at leastone cysteine residue. Alternatively, the component of the polymericcarrier may be modified accordingly with a —SH moiety, preferably via achemical reaction with a compound carrying a—SH moiety, such that eachof the components of the polymeric carrier carries at least one such—SHmoiety. Such a compound carrying a —SH moiety may be e.g. an(additional) cysteine or any further (modified) amino acid or compoundof the component of the polymeric carrier, which carries a —SH moiety.Such a compound may also be any non-amino compound or moiety, whichcontains or allows to introduce a —SH moiety into the component asdefined herein. Such non-amino compounds may be attached to thecomponent of the polymeric carrier according to the present inventionvia chemical reactions or binding of compounds, e.g. by binding of a3-thio propionic acid or 2-iminothiolane (Traut's reagent), by amideformation (e.g. carboxylic acids, sulphonic acids, amines, etc.), byMichael addition (e.g maleimide moieties, α,β unsaturated carbonyls,etc.), by click chemistry (e.g. azides or alkines), by alkene/alkinemethathesis (e.g. alkenes or alkines), imine or hydrozone formation(aldehydes or ketones, hydrazines, hydroxylamines, amines), complexationreactions (avidin, biotin, protein G) or components which allow Se-typesubstitution reactions (e.g halogenalkans, thiols, alcohols, amines,hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) orother chemical moieties which can be utilized in the attachment offurther components. In some cases the —SH moiety may be masked byprotecting groups during chemical attachment to the component. Suchprotecting groups are known in the art and may be removed after chemicalcoupling. In each case, the —SH moiety, e.g. of a cysteine or of anyfurther (modified) amino acid or compound, may be present at theterminal ends or internally at any position of the component of thepolymeric carrier. As defined herein, each of the components of thepolymeric carrier typically exhibits at least one —SH-moiety, but mayalso contain two, three, four, five, or even more —SH-moieties.Additionally to binding of cationic components a —SH moiety may be usedto attach further components of the polymeric carrier as defined herein,particularly an amino acid component, e.g. antigen epitopes, antigens,antibodies, cell penetrating peptides (e.g. TAT), ligands, etc.

As defined above, the polymeric carrier of the inventive polymericcarrier cargo molecule is formed by disulfide-crosslinked cationic (orpolycationic) components.

According to one first alternative, at least one cationic (orpolycationic) component of the polymeric carrier may be selected fromcationic or polycationic peptides or proteins. Such cationic orpolycationic peptides or proteins preferably exhibit a length of about 3to 100 amino acids, preferably a length of about 3 to 50 amino acids,more preferably a length of about 3 to 25 amino acids, e.g. a length ofabout 3 to 10; 5 to 20; 5 to 15; 8 to 15, 16 or 17; 10 to 15, 16, 17,18, 19, or 20; or 15 to 25 amino acids. Alternatively or additionally,such cationic or polycationic peptides or proteins may exhibit amolecular weight of about 0.01 kDa to about 100 kDa, including amolecular weight of about 0.5 kDa to about 100 kDa, preferably of about10 kDa to about 50 kDa, even more preferably of about 10 kDa to about 30kDa.

In the specific case that the cationic component of the polymericcarrier comprises a cationic or polycationic peptide or protein, thecationic properties of the cationic or polycationic peptide or proteinor of the entire polymeric carrier, if the polymeric carrier is entirelycomposed of cationic or polycationic peptides or proteins, may bedetermined upon its content of cationic amino acids. Preferably, thecontent of cationic amino acids in the cationic or polycationic peptideor protein and/or the polymeric carrier is at least 10%, 20%, or 30%,preferably at least 40%, more preferably at least 50%, 60% or 70%, butalso preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to 90%,more preferably in the range of about 15% to 75%, even more preferablyin the range of about 20% to 50%, e.g. 20, 30, 40 or 50%, or in a rangeformed by any two of the afore mentioned values, provided, that thecontent of all amino acids, e.g. cationic, lipophilic, hydrophilic,aromatic and further amino acids, in the cationic or polycationicpeptide or protein, or in the entire polymeric carrier, if the polymericcarrier is entirely composed of cationic or polycationic peptides orproteins, is 100%.

In this context, cationic amino acids are preferably the naturallyoccurring amino acids Arg (Arginine), Lys (Lys₁ ne), His (Histidine),and Orn (Ornithin). However, in a broader sense any (non-natural) aminoacid carrying a cationic charg on its side chain may also be envisagedto carry out the invention. Preferably, however, are those cationicamino acids, the side chains of which are positively charged underphysiological pH conditions. In a more preferred embodiment, these aminoacids are Arg, Lys, and Orn.

Preferably, such cationic or polycationic peptides or proteins of thepolymeric carrier, which comprise or are additionally modified tocomprise at least one —SH moeity, are selected from, without beingrestricted thereto, cationic peptides or proteins such as protamine,nucleolin, spermine or spermidine, oligo- or poly-L-lysine (PLL), basicpolypeptides, oligo or poly-arginine, cell penetrating peptides (CPPs),chimeric CPPs, such as Transportan, or MPG peptides, HIV-bindingpeptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, members of thepenetratin family, e.g. Penetratin, Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, pIsl, etc.,antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, SynB, SynB(1),pVEC, hCT-derived peptides, SAP, MAP, KALA, PpTG20, Loligomere, FGF,Lactoferrin, histones, VP22 derived or analog peptides, HSV, VP22(Herpes simplex), MAP, KALA or protein transduction domains (PTDs,PpT620, prolin-rich peptides, arginine-rich peptides, lysine-richpeptides, Pep-1, L-oligomers, Calcitonin peptide(s), etc.

Alternatively or additionally, such cationic or polycationic peptides orproteins of the polymeric carrier, which comprise or are additionallymodified to comprise at least one —SH moeity, are selected from, withoutbeing restricted thereto, following cationic peptides having thefollowing sum formula (I):

{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)};

wherein l+m+n+o+x=3-100, and l, m, n or o independently of each other isany number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80,81-90 and 91-100 provided that the overall content of Arg (Arginine),Lys (Lys₁ ne), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide; and Xaa is any amino acidselected from native (=naturally occurring) or non-native amino acidsexcept of Arg, Lys, His or Orn; and x is any number selected from 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, provided, that theoverall content of Xaa does not exceed 90% of all amino acids of theoligopeptide. Any of amino acids Arg, Lys, His, Orn and Xaa may bepositioned at any place of the peptide. In this context cationicpeptides or proteins in the range of 7-30 amino acids are particularpreferred. Even more preferred peptides of this formula areoligoarginines such as e.g. Arg₇ (SEQ ID NO: 392), Arg₈ (SEQ ID NO:393), Arg₉ (SEQ ID NO:), Arg₁₂ (SEQ ID NO: 397), His₃Arg₉ (SEQ ID NO:428), Arg₉His₃ (SEQ ID NO: 429), His₃Arg₉His₃ (SEQ ID NO: 430),His₆Arg₉His₆ (SEQ ID NO: 431), His₃Arg₄His₃ (SEQ ID NO: 432),His₆Arg₄His₆ (SEQ ID NO: 433), TyrSer₂Arg₉Ser₂Tyr (SEQ ID NO: 434),(ArgLysHis)₄ (SEQ ID NO: 435), Tyr(ArgLysHis)₂Arg (SEQ ID NO: 436), etc.

According to a particular preferred embodiment, such cationic orpolycationic peptides or proteins of the polymeric carrier having theempirical sum formula (I) as shown above, may, without being restrictedthereto, comprise at least one of the following subgroup of formulae:

-   -   Arg₇ (SEQ ID NO: 392), Arg₈ (SEQ ID NO: 393), Arg₉ (SEQ ID NO:        394), Arg₁₀ (SEQ ID NO: 395), Arg₁₁ (SEQ ID NO: 396), Arg₁₂ (SEQ        ID NO: 397), Arg₁₃ (SEQ ID NO: 398), Arg₁₄ (SEQ ID NO: 399),        Arg₁₅₋₃₀ (SEQ ID NO: 400);    -   Lys₇ (SEQ ID NO: 401), Lys₈ (SEQ ID NO: 402), Lys₉ (SEQ ID NO:        403), Lys₁₀ (SEQ ID NO: 404), Lys₁₁ (SEQ ID NO: 405), Lys₁₂ (SEQ        ID NO: 406), Lys₁₃ (SEQ ID NO: 407), Lys₁₄ (SEQ ID NO: 408),        Lys₁₅₋₃₀ (SEQ ID NO: 409);    -   His₇ (SEQ ID NO: 410), His₈ (SEQ ID NO: 411), His₉ (SEQ ID NO:        412), His₁₀ (SEQ ID NO: 413), His₁₁ (SEQ ID NO: 414), His₁₂ (SEQ        ID NO: 415), His₁₃ (SEQ ID NO: 416), His₁₄ (SEQ ID NO: 417),        His₁₅₋₃₀ (SEQ ID NO: 418);    -   Orn₇ (SEQ ID NO: 419), Orn₈ (SEQ ID NO: 420), Orn₉ (SEQ ID NO:        421), Orn₁₀ (SEQ ID NO: 422), Orn₁₁ (SEQ ID NO: 423), Orn₁₂ (SEQ        ID NO: 424), Orn₁₃ (SEQ ID NO: 425), Orn₁₄ (SEQ ID NO: 426),        Orn₁₅₋₃₀ (SEQ ID NO: 427);

According to a further particularly preferred embodiment, cationic orpolycationic peptides or proteins of the polymeric carrier, having theempirical sum formula (I) as shown above and which comprise or areadditionally modified to comprise at least one —SH moeity, may bepreferably selected from, without being restricted thereto, at least oneof the following subgroup of formulae. The following formulae (as withempirical formula (I)) do not specify any amino acid order, but areintended to reflect empirical formulae by exclusively specifying the(number of) amino acids as components of the respective peptide.Accordingly, as an example, empirical formula Arg₍₇₋₂₉₎Lys₁ (SEQ ID NO:437) is intended to mean that peptides falling under this formulacontain 7 to 19 Arg residues and 1 Lys residue of whatsoever order. Ifthe peptides contain 7 Arg residues and 1 Lys residue, all variantshaving 7 Arg residues and 1 Lys residue are encompassed. The Lys residuemay therefore be positioned anywhere in the e.g. 8 amino acid longsequence composed of 7 Arg and 1 Lys residues. The subgroup preferablycomprises:

-   -   Arg₍₄₋₂₉₎Lys₁ (SEQ ID NO: 441), Arg₍₄₋₂₉₎His₁ (SEQ ID NO: 442),        Arg₍₄₋₂₉Orn₁ (SEQ ID NO: 443), Lys_((4_29))His₁ (SEQ ID NO:        444), Lys_((4_29))Orn₁ (SEQ ID NO: 445), His₍₄₋₂₉₎Orn₁ (SEQ ID        NO: 446),    -   Arg₍₃₋₂₈₎Lys₂ (SEQ ID NO: 447), Arg₍₃₋₂₈₎His₂ (SEQ ID NO: 448),        Arg₍₃₋₂₈₎Orn₂ (SEQ ID NO: 449), Lys₍₃₋₂₈₎His₂ (SEQ ID NO: 450),        Lys₍₃₋₂₈₎Orn₂ (SEQ ID NO: 451), His₍₃₋₂₈₎Orn₂ (SEQ ID NO: 452),    -   Arg₍₂₋₂₇₎Lys₃ (SEQ ID NO: 453), Arg₍₂₋₂₇₎His₃ (SEQ ID NO: 454),        Arg₍₂₋₂₇₎Orn₃ (SEQ ID NO: 455), Lys₍₂₋₂₇₎His₃ (SEQ ID NO: 456),        Lys₍₂₋₂₇₎Orn₃ (SEQ ID NO: 457), His₍₂₋₂₇₎Orn₃ (SEQ ID NO: 458),    -   Arg₍₁₋₂₆₎Lys₄ (SEQ ID NO: 459), Arg₍₁₋₂₆₎His₄ (SEQ ID NO: 460),        Arg₍₁₋₂₆₎Orn₄ (SEQ ID NO: 461), Lys₍₁₋₂₆₎His₄ (SEQ ID NO: 462),        Lys₍₁₋₂₆₎Orn₄ (SEQ ID NO: 463), His₍₁₋₂₆₎Orn₄ (SEQ ID NO: 464),    -   Arg₍₃₋₂₈₎Lys₁His₁ (SEQ ID NO: 465), Arg₍₃₋₂₈₎Lys₁Orn₁ (SEQ ID        NO: 466), Arg₍₃₋₂₈₎His₁Orn₁ (SEQ ID NO: 467), Arg₁Lys₍₃₋₂₈₎His₁        (SEQ ID NO: 468), Arg₁Lys₍₃₋₂₈₎Orn₁ (SEQ ID NO: 469),        Lys₍₃₋₂₈₎His₁Orn₁ (SEQ ID NO: 470), Arg₁Lys₁His₍₃₋₂₈₎ (SEQ ID        NO: 471), Arg₁His₍₃₋₂₈₎Orn₁ (SEQ ID NO: 472), Lys₁His₍₃₋₂₈₎Orn₁        (SEQ ID NO: 473);    -   Arg₍₂₋₂₇₎Lys₂His₁ (SEQ ID NO: 474), Arg₍₂₋₂₇₎Lys₁His₂ (SEQ ID        NO: 475), Arg₍₂₋₂₇₎Lys₂Orn₁ (SEQ ID NO: 476), Arg₍₂₋₂₇₎Lys₁Orn₂        (SEQ ID NO: 477), Arg₍₂₋₂₇₎His₂Orn₁ (SEQ ID NO: 478),        Arg₍₂₋₂₇₎His₁Orn₂ (SEQ ID NO: 479), Arg₂Lys₍₂₋₂₇₎His₁ (SEQ ID        NO: 480), Arg₁Lys(2-27)His₂ (SEQ ID NO: 481), Arg₂Lys₍₂₋₂₇₎Orn₁        (SEQ ID NO: 482), Arg₁Lys₍₂₋₂₇₎Orn₂ (SEQ ID NO: 483),        Lys₍₂₋₂₇₎His₂Orn₁ (SEQ ID NO: 484), Lys₍₂₋₂₇₎His₁Orn₂ (SEQ ID        NO: 485), Arg₂Lys₁His₍₂₋₂₇₎ (SEQ ID NO: 486), Arg₁Lys₂His₍₂₋₂₇₎        (SEQ ID NO: 487), Arg₂His₍₂₋₂₇₎Orn₁ (SEQ ID NO: 488),        Arg₁His₍₂₋₂₇₎Orn₂ (SEQ ID NO: 489), Lys₂His₍₂₋₂₇₎Orn₁ (SEQ ID        NO: 490), Lys₁His₍₂₋₂₇₎Orn₂ (SEQ ID NO: 491);    -   Arg₍₁₋₂₆₎Lys₃His₁ (SEQ ID NO: 492), Arg₍₁₋₂₆₎Lys₂His₂ (SEQ ID        NO: 493), Arg₍₁₋₂₆₎Lys₁His₃ (SEQ ID NO: 494), Arg₍₁₋₂₆₎Lys₃Orn₁        (SEQ ID NO: 495), Arg₍₁₋₂₆₎Lys₂Orn₂ (SEQ ID NO: 496),        Arg₍₁₋₂₆₎Lys₁Orn₃ (SEQ ID NO: 497), Arg₍₁₋₂₆₎His₃Orn₁ (SEQ ID        NO: 498), Arg₍₁₋₂₆₎His₂Orn₂ (SEQ ID NO: 499), Arg₍₁₋₂₆₎His₁Orn₃        (SEQ ID NO: 500), Arg₃Lys₍₁₋₂₆₎His₁ (SEQ ID NO: 501),    -   Arg₂Lys₍₁₋₂₆₎His₂ (SEQ ID NO: 502), Arg₁Lys₍₁₋₂₆₎His₃ (SEQ ID        NO: 503), Arg₃Lys₍₁₋₂₆₎Orn₁ (SEQ ID NO: 504), Arg₂Lys₍₁₋₂₆₎Orn₂        (SEQ ID NO: 505), Arg₁Lys₍₁₋₂₆₎Orn₃ (SEQ ID NO: 506),        Lys₍₁₋₂₆₎His₃Orn₁ (SEQ ID NO: 507), Lys₍₁₋₂₆₎His₂Orn₂ (SEQ ID        NO: 508), Lys₍₁₋₂₆₎His₁Orn₃ (SEQ ID NO: 509), Arg₃Lys₁His₍₁₋₂₆₎        (SEQ ID NO: 510), Arg₂Lys₂His₍₁₋₂₆₎ (SEQ ID NO: 511),        Arg₁Lys₃His₍₁₋₂₆₎ (SEQ ID NO: 512), Arg₃His₍₁₋₂₆₎Orn₁ (SEQ ID        NO: 513), Arg₂His₍₁₋₂₆₎Orn₂ (SEQ ID NO: 514), Arg₁His₍₁₋₂₆₎Orn₃        (SEQ ID NO: 515), Lys₃His₍₁₋₂₆₎Orn₁ (SEQ ID NO: 516),        Lys₂His₍₁₋₂₆₎Orn₂ (SEQ ID NO: 517), Lys₁His₍₁₋₂₆₎Orn₃ (SEQ ID        NO: 518);    -   Arg₍₂₋₂₇₎Lys₁His₁Orn₁ (SEQ ID NO: 519), Arg₁Lys₍₂₋₂₇₎His₁Orn₁        (SEQ ID NO: 520), Arg₁Lys₁His₍₂₋₂₇₎Orn₁ (SEQ ID NO: 521),        Arg₁Lys₁His₁Orn₍₂₋₂₇₎ (SEQ ID NO: 522);    -   Arg₍₁₋₂₆₎Lys₂His₁Orn₁ (SEQ ID NO: 523), Arg₍₁₋₂₆₎Lys₁His₂Orn₁        (SEQ ID NO: 524), Arg₍₁₋₂₆₎Lys₁His₁Orn₂ (SEQ ID NO: 525),        Arg₂Lys₍₁₋₂₆₎His₁Orn₁ (SEQ ID NO: 526), Arg₁Lys₍₁₋₂₆₎His₂Orn₁        (SEQ ID NO: 527), Arg₁Lys₍₁₋₂₆₎His₁Orn₂ (SEQ ID NO: 528),        Arg₂Lys₁His₍₁₋₂₆₎Orn₁ (SEQ ID NO: 529), Arg₁Lys₂His₍₁₋₂₆₎Orn₁        (SEQ ID NO: 530), Arg₁Lys₁His₍₁₋₂₆₎Orn₂ (SEQ ID NO: 531),        Arg₂Lys₁His₁Orn₍₁₋₂₆₎ (SEQ ID NO: 532), Arg₁Lys₂His₁Orn₍₁₋₂₆₎        (SEQ ID NO: 533), Arg₁Lys₁His₂Orn₍₁₋₂₆₎ (SEQ ID NO: 534);

According to a further particular preferred embodiment, cationic orpolycationic peptides or proteins of the polymeric carrier, having theempirical sum formula (I) as shown above and which comprise or areadditionally modified to comprise at least one —SH moeity, may be,without being restricted thereto, selected from the subgroup consistingof generic formulas Arg₇ (also termed as R₇; SEQ ID NO: 392), Arg₉ (alsotermed R₉; SEQ ID NO: 394), Arg₁₂ (also termed as R₁₂; SEQ ID NO: 397).

According to a one further particular preferred embodiment, the cationicor polycationic peptide or protein of the polymeric carrier, whendefined according to formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (I)) asshown above and which comprise or are additionally modified to compriseat least one —SH moeity, may be, without being restricted thereto,selected from subformula (Ia):

{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa′)_(x)}  formula (Ia)

wherein (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o); and x are as definedherein, Xaa′ is any amino acid selected from native (=naturallyoccurring) or non-native amino acids except of Arg, Lys, His, Orn or Cysand y is any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70,71-80 and 81-90, provided that the overall content of Arg (Arginine),Lys (Lysine), His (Histidine) and Orn (Ornithine) represents at least10% of all amino acids of the oligopeptide.

This embodiment may apply to situations, wherein the cationic orpolycationic peptide or protein of the polymeric carrier, e.g. whendefined according to empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula (I)) as shownabove, comprises or has been modified with at least one cysteine as —SHmoiety in the above meaning such that the cationic or polycationicpeptide as cationic component carries at least one cysteine, which iscapable to form a disulfide bond with other components of the polymericcarrier.

According to another particular preferred embodiment, the cationic orpolycationic peptide or protein of the polymeric carrier, when definedaccording to formula {(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}(formula (I)) as shown above, may be, without being restricted thereto,selected from subformula (Ib):

Cys₁{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}Cys₂  (formula(Ib))

wherein empirical formula{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)} (formula (I)) is asdefined herein and forms a core of an amino acid sequence according to(semiempirical) formula (I) and wherein Cys₁ and Cys₂ are Cysteinesproximal to, or terminal to(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x). Exemplary examplesmay comprise any of the above sequences flanked by two Cys and followingsequences:

(SEQ ID NOs: 1-14): Cys(Arg₇)Cys, Cys(Arg₈)Cys, Cys(Arg₉)Cys,Cys(Arg₁₀)Cys, Cys(Arg₁₁)Cys, Cys(Arg₁₂)Cys,Cys(Arg₁₃)Cys, Cys(Arg₁₄)Cys, Cys(Arg₁₅)Cys,Cys(Arg₁₆)Cys, Cys(Arg₁₇)Cys, Cys(Arg₁₈)Cys,Cys(Arg₁₉)Cys, Cys(Arg₂₀)Cys CysArg₇Cys (SEQ ID NO. 1)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₈Cys (SEQ ID NO. 2)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₉Cys: (SEQ ID NO. 3)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₁₀Cys (SEQ ID NO. 4)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₁₁Cys(SEQ ID NO. 5) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-CysCysArg₁₂Cys: (SEQ ID NO. 6)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Cys CysArg₁₃Cys:(SEQ ID NO. 7) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₁₄Cys: (SEQ ID NO. 8)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-CysCysArg₁₅Cys: (SEQ ID NO. 9)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-Arg-CysCysArg₁₆Cys: (SEQ ID NO. 10)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Arg-Arg-Arg-Arg-Arg-CysCysArg₁₇Cys: (SEQ ID NO. 11)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₁₈Cys: (SEQ ID NO. 12)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₁₉Cys: (SEQ ID NO. 13)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys CysArg₂₀Cys: (SEQ ID NO. 14)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg Cys

This embodiment may apply to situations, wherein the cationic orpolycationic peptide or protein of the polymeric carrier, e.g. whendefined according to empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula (I)) as shownabove, has been modified with at least two cysteines as —SH moieties inthe above meaning such that the cationic or polycationic peptide of theinventive polymeric carrier cargo complex as cationic component carriesat least two (terminal) cysteines, which are capable to form a disulfidebond with other components of the polymeric carrier.

According to a second alternative, at least one cationic (orpolycationic) component of the polymeric carrier may be selected frome.g. any (non-peptidic) cationic or polycationic polymer suitable inthis context, provided that this (non-peptidic) cationic or polycationicpolymer exhibits or is modified to exhibit at least one —SH-moiety,which provide for a disulfide bond linking the cationic or polycationicpolymer with another component of the polymeric carrier as definedherein. Thus, likewise as defined herein, the polymeric carrier maycomprise the same or different cationic or polycationic polymers.

In the specific case that the cationic component of the polymericcarrier comprises a (non-peptidic) cationic or polycationic polymer thecationic properties of the (non-peptidic) cationic or polycationicpolymer may be determined upon its content of cationic charges whencompared to the overall charges of the components of the cationicpolymer. Preferably, the content of cationic charges in the cationicpolymer at a (physiological) pH as defined herein is at least 10%, 20%,or 30%, preferably at least 40%, more preferably at least 50%, 60% or70%, but also preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%,99% or 100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to90%, more preferably in the range of about 30% to 100%, even preferablyin the range of about 50% to 100%, e.g. 50, 60, 70, 80%, 90% or 100%, orin a range formed by any two of the afore mentioned values, provided,that the content of all charges, e.g. positive and negative charges at a(physiological) pH as defined herein, in the entire cationic polymer is100%.

Preferably, the (non-peptidic) cationic component of the polymericcarrier represents a cationic or polycationic polymer, typicallyexhibiting a molecular weight of about 0.1 or 0.5 kDa to about 100 kDa,preferably of about 1 kDa to about 75 kDa, more preferably of about 5kDa to about 50 kDa, even more preferably of about 5 kDa to about 30kDa, or a molecular weight of about 10 kDa to about 50 kDa, even morepreferably of about 10 kDa to about 30 kDa. Additionally, the(non-peptidic) cationic or polycationic polymer typically exhibits atleast one —SH-moiety, which is capable to form a disulfide linkage uponcondensation with either other cationic components or other componentsof the polymeric carrier as defined herein.

In the above context, the (non-peptidic) cationic component of thepolymeric carrier may be selected from acrylates, modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), chitosanes,aziridines or 2-ethyl-2-oxazoline (forming oligo ethylenimines ormodified oligoethylenimines), polymers obtained by reaction ofbisacrylates with amines forming oligo beta aminoesters or poly amidoamines, or other polymers like polyesters, polycarbonates, etc. Eachmolecule of these (non-peptidic) cationic or polycationic polymerstypically exhibits at least one —SH-moiety, wherein these at least one—SH-moiety may be introduced into the (non-peptidic) cationic orpolycationic polymer by chemical modifications, e.g. using imonothiolan,3-thio propionic acid or introduction of —SH-moieties containing aminoacids, such as cysteine or any further (modified) amino acid. Such—SH-moieties are preferably as already defined above.

In the context of the polymeric carrier, the cationic components, whichform basis for the polymeric carrier by disulfide-crosslinkage, may bethe same or different from each other. It is also particularly preferredthat the polymeric carrier of the present invention comprises mixturesof cationic peptides, proteins or polymers and optionally furthercomponents as defined herein, which are crosslinked by disulfide bondsas described herein.

In this context, the inventive polymeric carrier cargo complex due toits variable polymeric carrier advantageously allows to combine desiredproperties of different (short) cationic or polycationic peptides,proteins or polymers or other components. The polymeric carrier, e.g.,allows to efficiently compact nucleic acids for the purpose of efficienttransfection of nucleic acids, for adjuvant therapy, for the purposes ofgene therapy, for gene knock-down or others strategies without loss ofactivity, particularly exhibiting an efficient transfection of a nucleicacid into different cell lines in vitro but particularly transfection invivo. The polymeric carrier and thus the inventive polymeric carriercargo complex is furthermore not toxic to cells, provides for efficientrelease of its nucleic acid cargo, is stable during lyophilization andis applicable as immunostimulating agent or adjuvant. Preferably, thepolymer carrier cargo complex may induce the anti-viral cytokineIFN-alpha.

In particular, the polymeric carrier formed by disulfide-linked cationiccomponents allows considerably to vary its peptide or polymeric contentand thus to modulate its biophysical/biochemical properties,particularly the cationic properties of the polymeric carrier, quiteeasily and fast, e.g. by incorporating as cationic components the sameor different cationic peptide(s) or polymer(s) and optionally addingother components into the polymeric carrier. Even though consisting ofquite small non-toxic monomer units the polymeric carrier forms a longcationic binding sequence providing a strong condensation of the nucleicacid cargo and complex stability. Under the reducing conditions of thecytosole (e.g. cytosolic GSH), the complex is rapidly degraded into its(cationic) components, which are further degraded (e.g. oligopeptides).This supports deliberation of the nucleic acid cargo in the cytosol. Dueto degradation into small oligopeptides or polymers in the cytosol, notoxicity is observed as known for high-molecular oligopeptides orpolymers, e.g. from high-molecular polyarginine.

Accordingly, the polymeric carrier of the inventive polymeric carriercargo complex may comprise different (short) cationic or polycationicpeptides, proteins or polymers selected from cationic or polycationicpeptides, proteins or (non-peptidic) polymers as defined above,optionally together with further components as defined herein.

Additionally, the polymeric carrier of the inventive polymeric carriercargo complex as defined above, more preferably at least one of thedifferent (short) cationic or polycationic peptides or (non-peptidic)polymers forming basis for the polymeric carrier viadisulfide-crosslinking, may be, preferably prior to thedisulfide-crosslinking, be modified with at least one further component.Alternatively, the polymeric carrier as such may be modified with atleast one further component. It may also optionally comprise at leastone further component, which typically forms the polymeric carrierdisulfide together with the other the (short) cationic or polycationicpeptides as defined above via disulfide crosslinking.

To allow modification of a cationic or polycationic peptide or a(non-peptidic) polymer as defined above, each of the components of thepolymeric carrier may (preferably already prior todisulfide-crosslinking) also contain at least one further functionalmoiety, which allows attaching such further components as definedherein. Such functional moieties may be selected from functionalitieswhich allow the attachment of further components, e.g. functionalitiesas defined herein, e.g. by amide formation (e.g. carboxylic acids,sulphonic acids, amines, etc.), by Michael addition (e.g maleinimidemoieties, α,β unsatured carbonyls, etc.), by click chemistry (e.g.azides or alkines), by alkene/alkine methatesis (e.g. alkenes oralkines), imine or hydrozone formation (aldehydes or ketons, hydrazins,hydroxylamins, amines), complexation reactions (avidin, biotin, proteinG) or components which allow S_(n)-type substitution reactions (e.ghalogenalkans, thiols, alcohols, amines, hydrazines, hydrazides,sulphonic acid esters, oxyphosphonium salts) or other chemical moietieswhich can be utilized in the attachment of further components.

According to a particularly preferred embodiment, the further component,which may be contained in the polymeric carrier or which may be used tomodify the different (short) cationic or polycationic peptides or(non-peptidic) polymers forming basis for the polymeric carrier of theinventive polymeric carrier cargo complex is an amino acid component(AA), which may e.g. modify the biophysical/biochemical properties ofthe polymeric carrier as defined herein.

According to the present invention, the amino acid component (AA)comprises a number of amino acids preferably in a range of about 1 to100, preferably in a range of about 1 to 50, more preferably selectedfrom a number comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14or 15-20, or may be selected from a range formed by any two of the aforementioned values. In this context the amino acids of amino acidcomponent (AA) can be chosen independently from each other. For exampleif in the polymeric carrier two or more (AA) components are present theycan be the same or can be different from each other.

The amino acid component (AA) may contain or may be flanked (e.g.terminally) by a —SH containing moiety, which allows introducing thiscomponent (AA) via a disulfide bond into the polymeric carrier asdefined herein. In the specific case that the —SH containing moietyrepresents a cysteine, the amino acid component (AA) may also be read as-Cys-(AA)-Cys- wherein Cys represents Cysteine and provides for thenecessary —SH-moiety for a disulfide bond. The —SH containing moiety maybe also introduced into amino acid component (AA) using any ofmodifications or reactions as shown above for the cationic component orany of its components.

Furthermore, the amino acid component (AA) may be provided with two—SH-moieties (or even more), e.g. in a form represented by formulaHS-(AA)-SH to allow binding to two functionalities via disulfide bonds,e.g. if the amino acid component (AA) is used as a linker between twofurther components (e.g. as a linker between two cationic polymers). Inthis case, one —SH moiety is preferably protected in a first step usinga protecting group as known in the art, leading to an amino acidcomponent (AA) of formula HS-(AA)-S-protecting group. Then, the aminoacid component (AA) may be bound to a further component of the polymericcarrier, to form a first disulfide bond via the non-protected —SHmoiety. The protected —SH-moiety is then typically deprotected and boundto a further free —SH-moiety of a further component of the polymericcarrier to form a second disulfide bond.

Alternatively, the amino acid component (AA) may be provided with otherfunctionalities as already described above for the other components ofthe polymeric carrier, which allow binding of the amino acid component(AA) to any of components of the polymeric carrier.

Thus, according to the present invention, the amino acid component (AA)may be bound to further components of the polymeric carrier with orwithout using a disulfide linkage. Binding without using a disulfidelinkage may be accomplished by any of the reactions described above,preferably by binding the amino acid component (AA) to the othercomponent of the polymeric carrier using an amid-chemistry as definedherein. If desired or necessary, the other terminus of the amino acidcomponent (AA), e.g. the N- or C-terminus, may be used to couple anothercomponent, e.g. a ligand L. For this purpose, the other terminus of theamino acid component (AA) preferably comprises or is modified tocomprise a further functionality, e.g. an alkyn-species (see above),which may be used to add the other component via e.g. click-chemistry.If the ligand is bound via an acid-labile bond, the bond is preferablycleaved off in the endosome and the polymeric carrier presents aminoacid component (AA) at its surface.

The amino acid component (AA) may occur as a further component of thepolymeric carrier as defined above, e.g. as a linker between cationiccomponents e.g. as a linker between one cationic peptide and a furthercationic peptide, as a linker between one cationic polymer and a furthercationic polymer, as a linker between one cationic peptide and acationic polymer, all preferably as defined herein, or as an additionalcomponent of the polymeric carrier, e.g. by binding the amino acidcomponent (AA) to the polymeric carrier or a component thereof, e.g. viaside chains, SH-moieties or via further moieties as defined herein,wherein the amino acid component (AA) is preferably accordinglymodified.

According to a further and particularly preferred alternative, the aminoacid component (AA), may be used to modify the polymeric carrier,particularly the content of cationic components in the polymeric carrieras defined above.

In this context it is preferable, that the content of cationiccomponents in the polymeric carrier is at least 10%, 20%, or 30%,preferably at least 40%, more preferably at least 50%, 60% or 70%, butalso preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99% or 100%, or may be in the range of about 30% to 100%,more preferably in the range of about 50% to 100%, even preferably inthe range of about 70% to 100%, e.g. 70, 80, 90 or 100%, or in a rangeformed by any two of the afore mentioned values, provided, that thecontent of all components in the polymeric carrier is 100%.

In the context of the present invention, the amino acid component (AA)may be selected from the following alternatives.

According to a first alternative, the amino acid component (AA) may bean aromatic amino acid component (AA). The incorporation of aromaticamino acids or sequences as amino aromatic acid component (AA) into thepolymeric carrier of the present invention enables a different (second)binding of the polymeric carrier to the nucleic acid due to interactionsof the aromatic amino acids with the bases of the nucleic acid cargo incontrast to the binding thereof by cationic charged sequences of thepolymeric carrier molecule to the phosphate backbone. This interactionmay occur e.g. by intercalations or by minor or major groove binding.This kind of interaction is not prone to decompaction by anioniccomplexing partners (e.g. Heparin, Hyaluronic acids) which are foundmainly in the extracellular matrix in vivo and is also less susceptibleto salt effects.

For this purpose, the amino acids in the aromatic amino acid component(AA) may be selected from either the same or different aromatic aminoacids e.g. selected from Trp, Tyr, or Phe. Alternatively, the aminoacids (or the entire aromatic amino acid component (AA)) may be selectedfrom following peptide combinations Trp-Tyr, Tyr-Trp, Trp-Trp, Tyr-Tyr,Trp-Tyr-Trp, Tyr-Trp-Tyr, Trp-Trp-Trp, Tyr-Tyr-Tyr, Trp-Tyr-Trp-Tyr,Tyr-Trp-Tyr-Trp, Trp-Trp-Trp-Trp, Phe-Tyr, Tyr-Phe, Phe-Phe,Phe-Tyr-Phe, Tyr-Phe-Tyr, Phe-Phe-Phe, Phe-Tyr-Phe-Tyr, Tyr-Phe-Tyr-Phe,Phe-Phe-Phe-Phe, Phe-Trp, Trp-Phe, Phe-Phe, Phe-Trp-Phe, Trp-Phe-Trp,Phe-Trp-Phe-Trp, Trp-Phe-Trp-Phe, or Tyr-Tyr-Tyr-Tyr, etc. (SEQ ID NOs:15-42). Such peptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 12, 13, 14, 15 or even more times. These peptidecombinations may also be combined with each other as suitable.

Additionally, the aromatic amino acid component (AA) may contain or maybe flanked by a —SH containing moiety, which allows introducing thiscomponent via a disulfide bond as a further part of the polymericcarrier as defined above, e.g. as a linker. Such a —SH containing moietymay be any moiety as defined herein suitable to couple one component asdefined herein to a further component as defined herein. As an example,such a —SH containing moiety may be a cysteine. Then, e.g. the aromaticamino acid component (AA) may be selected from e.g. peptide combinationsCys-Tyr-Cys, Cys-Trp-Cys, Cys-Trp-Tyr-Cys, Cys-Tyr-Trp-Cys,Cys-Trp-Trp-Cys, Cys-Tyr-Tyr-Cys, Cys-Trp-Tyr-Trp-Cys,Cys-Tyr-Trp-Tyr-Cys, Cys-Trp-Trp-Trp-Cys, Cys-Tyr-Tyr-Tyr-Cys,Cys-Trp-Tyr-Trp-Tyr-Cys, Cys-Tyr-Trp-Tyr-Trp-Cys,Cys-Trp-Trp-Trp-Trp-Cys, Cys-Tyr-Tyr-Tyr-Tyr-Cys, Cys-Phe-Cys,Cys-Phe-Tyr-Cys, Cys-Tyr-Phe-Cys, Cys-Phe-Phe-Cys, Cys-Tyr-Tyr-Cys,Cys-Phe-Tyr-Phe-Cys, Cys-Tyr-Phe-Tyr-Cys, Cys-Phe-Phe-Phe-Cys,Cys-Tyr-Tyr-Tyr-Cys, Cys-Phe-Tyr-Phe-Tyr-Cys, Cys-Tyr-Phe-Tyr-Phe-Cys,or Cys-Phe-Phe-Phe-Phe-Cys, Cys-Phe-Trp-Cys, Cys-Trp-Phe-Cys,Cys-Phe-Phe-Cys, Cys-Phe-Trp-Phe-Cys, Cys-Trp-Phe-Trp-Cys,Cys-Phe-Trp-Phe-Trp-Cys, Cys-Trp-Phe-Trp-Phe-Cys, etc. (SEQ ID NOs:43-75). Each Cys above may also be replaced by any modified peptide orchemical compound carrying a free —SH-moiety as defined herein. Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the aromatic amino acid component (AA) may contain orrepresent at least one proline, which may serve as a structure breakerof longer sequences of Trp, Tyr and Phe in the aromatic amino acidcomponent (AA), preferably two, three or more prolines.

According to a second alternative, the amino acid component (AA) may bea hydrophilic (and preferably non charged polar) amino acid component(AA). The incorporation of hydrophilic (and preferably non chargedpolar) amino acids or sequences as amino hydrophilic (and preferably noncharged polar) acid component (AA) into the polymeric carrier of thepresent invention enables a more flexible binding to the nucleic acidcargo. This leads to a more effective compaction of the nucleic acidcargo and hence to a better protection against nucleases and unwanteddecompaction. It also allows provision of a (long) polymeric carrierwhich exhibits a reduced cationic charge over the entire carrier and inthis context to better adjusted binding properties, if desired ornecessary.

For this purpose, the amino acids in the hydrophilic (and preferably noncharged polar) amino acid component (AA) may be selected from either thesame or different hydrophilic (and preferably non charged polar) aminoacids e.g. selected from Thr, Ser, Asn or Gln. Alternatively, the aminoacids (or the entire hydrophilic (and preferably non charged polar)amino acid component (AA)) may be selected from following peptidecombinations Ser-Thr, Thr-Ser, Ser-Ser, Thr-Thr, Ser-Thr-Ser,Thr-Ser-Thr, Ser-Ser-Ser, Thr-Thr-Thr, Ser-Thr-Ser-Thr, Thr-Ser-Thr-Ser,Ser-Ser-Ser-Ser, Thr-Thr-Thr-Thr, Gln-Asn, Asn-Gln, Gln-Gln, Asn-Asn,Gln-Asn-Gln, Asn-Gln-Asn, Gln-Gln-Gln, Asn-Asn-Asn, Gln-Asn-Gln-Asn,Asn-Gln-Asn-Gln, Gln-Gln-Gln-Gln, Asn-Asn-Asn-Asn, Ser-Asn, Asn-Ser,Ser-Ser, Asn-Asn, Ser-Asn-Ser, Asn-Ser-Asn, Ser-Ser-Ser, Asn-Asn-Asn,Ser-Asn-Ser-Asn, Asn-Ser-Asn-Ser, Ser-Ser-Ser-Ser, or Asn-Asn-Asn-Asn,etc. (SEQ ID NOs: 76-111) Such peptide combinations may be repeated e.g.1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15 or even more times. Thesepeptide combinations may also be combined with each other as suitable.

Additionally, the hydrophilic (and preferably non-charged polar) aminoacid component (AA) may contain or may be flanked by a —SH containingmoiety, which allows introducing this component via a disulfide bond asa further part of generic formula (I) above, e.g. as a linker. Such a—SH containing moiety may be any moiety as defined herein suitable tocouple one component as defined herein to a further component as definedherein. As an example, such a —SH containing moiety may be a cysteine.Then, e.g. the hydrophilic (and preferably non-charged polar) amino acidcomponent (AA) may be selected from e.g. peptide combinationsCys-Thr-Cys, Cys-Ser-Cys, Cys-Ser-Thr-Cys, Cys-Thr-Ser-Cys,Cys-Ser-Ser-Cys, Cys-Thr-Thr-Cys, Cys-Ser-Thr-Ser-Cys,Cys-Thr-Ser-Thr-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Thr-Thr-Thr-Cys,Cys-Ser-Thr-Ser-Thr-Cys, Cys-Thr-Ser-Thr-Ser-Cys,Cys-Ser-Ser-Ser-Ser-Cys, Cys-Thr-Thr-Thr-Thr-Cys, Cys-Asn-Cys,Cys-Gln-Cys, Cys-Gln-Asn-Cys, Cys-Asn-Gln-Cys, Cys-Gln-Gln-Cys,Cys-Asn-Asn-Cys, Cys-Gln-Asn-Gln-Cys, Cys-Asn-Gln-Asn-Cys,Cys-Gln-Gln-Gln-Cys, Cys-Asn-Asn-Asn-Cys, Cys-Gln-Asn-Gln-Asn-Cys,Cys-Asn-Gln-Asn-Gln-Cys, Cys-Gln-Gln-Gln-Gln-Cys,Cys-Asn-Asn-Asn-Asn-Cys, Cys-Asn-Cys, Cys-Ser-Cys, Cys-Ser-Asn-Cys,Cys-Asn-Ser-Cys, Cys-Ser-Ser-Cys, Cys-Asn-Asn-Cys, Cys-Ser-Asn-Ser-Cys,Cys-Asn-Ser-Asn-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Asn-Asn-Asn-Cys,Cys-Ser-Asn-Ser-Asn-Cys, Cys-Asn-Ser-Asn-Ser-Cys,Cys-Ser-Ser-Ser-Ser-Cys, or Cys-Asn-Asn-Asn-Asn-Cys, etc. (SEQ ID NOs:112-153). Each Cys above may also be replaced by any modified peptide orchemical compound carrying a free —SH-moiety as defined herein. Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the hydrophilic (and preferably non-charged polar) aminoacid component (AA) may contain at least one proline, which may serve asa structure breaker of longer sequences of Ser, Thr and Asn in thehydrophilic (and preferably non charged polar) amino acid component(AA), preferably two, three or more prolines.

According to a third alternative, the amino acid component (AA) may be alipohilic amino acid component (AA). The incorporation of lipohilicamino acids or sequences as amino lipohilic acid component (AA) into thepolymeric carrier of the present invention enables a stronger compactionof the nucleic acid cargo and/or the polymeric carrier and its nucleicacid cargo when forming a complex. This is particularly due tointeractions of one or more polymer strands of the polymeric carrier,particularly of lipophilic sections of lipohilic amino acid component(AA) and the nucleic acid cargo. This interaction will preferably add anadditional stability to the complex between the polymeric carrier andits nucleic acid cargo. This stabilization may somehow be compared to asort of non covalent crosslinking between different polymerstrands.Especially in aqueous environment this interaction is typically strongand provides a significant effect.

For this purpose, the amino acids in the lipophilic amino acid component(AA) may be selected from either the same or different lipophilic aminoacids e.g. selected from Leu, Val, Ile, Ala, Met. Alternatively, theamino acid AA (or the entire lipophilic amino acid component (AA)) maybe selected from following peptide combinations Leu-Val, Val-Leu,Leu-Leu, Val-Val, Leu-Val-Leu, Val-Leu-Val, Leu-Leu-Leu, Val-Val-Val,Leu-Val-Leu-Val, Val-Leu-Val-Leu, Leu-Leu-Leu-Leu, Val-Val-Val-Val,Ile-Ala, Ala-Ile, Ile-Ile, Ala-Ala, Ile-Ala-Ile, Ala-Ile-Ala,Ile-Ile-Ile, Ala-Ala-Ala, Ile-Ala-Ile-Ala, Ala-Ile-Ala-Ile,Ile-Ile-Ile-Ile, Ala-Ala-Ala-Ala, Met-Ala, Ala-Met, Met-Met, Ala-Ala,Met-Ala-Met, Ala-Met-Ala, Met-Met-Met, Ala-Ala-Ala, Met-Ala-Met-Ala,Ala-Met-Ala-Met, or Met-Met-Met-Met etc. (SEQ ID NOs: 154-188) Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the lipophilic amino acid component (AA) may contain ormay be flanked by a —SH containing moiety, which allows introducing thiscomponent via a disulfide bond as a further part of the polymericcarrier above, e.g. as a linker. Such a —SH containing moiety may be anymoiety as defined herein suitable to couple one component as definedherein to a further component as defined herein. As an example, such a—SH containing moiety may be a cysteine. Then, e.g. the lipophilic aminoacid component (AA) may be selected from e.g. peptide combinationsCys-Val-Cys, Cys-Leu-Cys, Cys-Leu-Val-Cys, Cys-Val-Leu-Cys,Cys-Leu-Leu-Cys, Cys-Val-Val-Cys, Cys-Leu-Val-Leu-Cys,Cys-Val-Leu-Val-Cys, Cys-Leu-Leu-Leu-Cys, Cys-Val-Val-Val-Cys,Cys-Leu-Val-Leu-Val-Cys, Cys-Val-Leu-Val-Leu-Cys,Cys-Leu-Leu-Leu-Leu-Cys, Cys-Val-Val-Val-Val-Cys, Cys-Ala-Cys,Cys-Ile-Cys, Cys-Ile-Ala-Cys, Cys-Ala-Ile-Cys, Cys-Ile-Ile-Cys,Cys-Ala-Ala-Cys, Cys-Ile-Ala-Ile-Cys, Cys-Ala-Ile-Ala-Cys,Cys-Ile-Ile-Ile-Cys, Cys-Ala-Ala-Ala-Cys, Cys-Ile-Ala-Ile-Ala-Cys,Cys-Ala-Ile-Ala-Ile-Cys, Cys-Ile-Ile-Ile-Ile-Cys, orCys-Ala-Ala-Ala-Ala-Cys, Cys-Met-Cys, Cys-Met-Ala-Cys, Cys-Ala-Met-Cys,Cys-Met-Met-Cys, Cys-Ala-Ala-Cys, Cys-Met-Ala-Met-Cys,Cys-Ala-Met-Ala-Cys, Cys-Met-Met-Met-Cys, Cys-Ala-Ala-Ala-Cys,Cys-Met-Ala-Met-Ala-Cys, Cys-Ala-Met-Ala-Met-Cys,Cys-Met-Met-Met-Met-Cys, or Cys-Ala-Ala-Ala-Ala-Cys, etc. (SEQ ID NOs:189-229).

Each Cys above may also be replaced by any modified peptide or chemicalcompound carrying a free —SH-moiety as defined herein. Such peptidecombinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13,14, 15 or even more times. These peptide combinations may also becombined with each other as suitable.

Additionally, the lipophilic amino acid component (AA) may contain atleast one proline, which may serve as a structure breaker of longersequences of Leu, Val, Ile, Ala and Met in the lipophilic amino acidcomponent (AA), preferably two, three or more prolines.

Finally, according to a fourth alternative, the amino acid component(AA) may be a weak basic amino acid component (AA). The incorporation ofweak basic amino acids or sequences as weak basic amino acid component(AA) into the polymeric carrier of the present invention may serve as aproton sponge and facilitates endosomal escape (also called endosomalrelease) (proton sponge effect). Incorporation of such a weak basicamino acid component (AA) preferably enhances transfection efficiency.

For this purpose, the amino acids in the weak basic amino acid component(AA) may be selected from either the same or different weak amino acidse.g. selected from histidine or aspartate (aspartic acid).Alternatively, the weak basic amino acids (or the entire weak basicamino acid component (AA)) may be selected from following peptidecombinations Asp-His, His-Asp, Asp-Asp, His-His, Asp-His-Asp,His-Asp-His, Asp-Asp-Asp, His-His-His, Asp-His-Asp-His, His-Asp-His-Asp,Asp-Asp-Asp-Asp, or His-His-His-His, etc. (SEQ ID NOs: 230-241) Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the weak basic amino acid component (AA) may contain ormay be flanked by a —SH containing moiety, which allows introducing thiscomponent via a disulfide bond as a further part of generic formula (I)above, e.g. as a linker. Such a —SH containing moiety may be any moietyas defined herein suitable to couple one component as defined herein toa further component as defined herein. As an example, such a —SHcontaining moiety may be a cysteine. Then, e.g. the weak basic aminoacid component (AA) may be selected from e.g. peptide combinationsCys-His-Cys, Cys-Asp-Cys, Cys-Asp-His-Cys, Cys-His-Asp-Cys,Cys-Asp-Asp-Cys, Cys-His-His-Cys, Cys-Asp-His-Asp-Cys,Cys-His-Asp-His-Cys, Cys-Asp-Asp-Asp-Cys, Cys-His-His-His-Cys,Cys-Asp-His-Asp-His-Cys, Cys-His-Asp-His-Asp-Cys,Cys-Asp-Asp-Asp-Asp-Cys, or Cys-His-His-His-His-Cys, etc. (SEQ ID NOs:242-255). Each Cys above may also be replaced by any modified peptide orchemical compound carrying a free —SH-moiety as defined herein. Suchpeptide combinations may be repeated e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 13, 14, 15 or even more times. These peptide combinations may alsobe combined with each other as suitable.

Additionally, the weak basic amino acid component (AA) may contain atleast one proline, which may serve as a structure breaker of longersequences of histidine or aspartate (aspartic acid) in the weak basicamino acid component (AA), preferably two, three or more prolines.

According to a fifth alternative, the amino acid component (AA) may be asignal peptide or signal sequence, a localization signal or sequence, anuclear localization signal or sequence (NLS), an antibody, a cellpenetrating peptide, (e.g. TAT), etc. Preferably such an amino acidcomponent (AA) is bound to the polymeric carrier or to another componentof the polymeric carrier via a (reversible) disulfide bond. In thiscontext the signal peptide or signal sequence, a localization signal orsequence, a nuclear localization signal or sequence (NLS), an antibody,a cell penetrating peptide, (e.g. TAT), etc.; additionally comprises atleast one —SH-moiety. In this context a signal peptide, a localizationsignal or sequence or a nuclear localization signal or sequence (NLS),may be used to direct the inventive polymeric carrier cargo complex tospecific target cells (e.g. hepatocytes or antigen-presenting cells) andpreferably allows a translocalization of the polymeric carrier to aspecific target, e.g. into the cell, into the nucleus, into theendosomal compartment, sequences for the mitochondrial matrix,localisation sequences for the plasma membrane, localisation sequencesfor the Golgi apparatus, the nucleus, the cytoplasm and thecytosceleton, etc. Such signal peptide, a localization signal orsequence or a nuclear localization signal may be used for the transportof any of the herein defined nucleic acids, preferably an RNA or a DNA,more preferably an shRNA or a pDNA, e.g. into the nucleus. Without beinglimited thereto, such a signal peptide, a localization signal orsequence or a nuclear localization signal may comprise, e.g.,localisation sequences for the endoplasmic reticulum. Particularlocalization signals or sequences or a nuclear localization signals mayinclude e.g. KDEL (SEQ ID NO: 256), DDEL (SEQ ID NO: 257), DEEL (SEQ IDNO: 258), QEDL (SEQ ID NO: 259), RDEL (SEQ ID NO: 260), and GQNLSTSN(SEQ ID NO: 261), nuclear localisation sequences, including PKKKRKV (SEQID NO: 262), PQKKIKS (SEQ ID NO: 263), QPKKP (SEQ ID NO: 264), RKKR (SEQID NO: 265), RKKRRQRRRAHQ (SEQ ID NO: 266), RQARRNRRRRWRERQR (SEQ ID NO:267), MPLTRRRPAASQALAPPTP (SEQ ID NO: 268), GAALTILV (SEQ ID NO: 269),and GAALTLLG (SEQ ID NO: 270), localisation sequences for the endosomalcompartment, including MDDQRDLISNNEQLP (SEQ ID NO: 271), localisationsequences for the mitochondrial matrix, includingMLFNLRXXLNNAAFRHGHNFMVRNFRCGQPLX (SEQ ID NO: 272), localisationsequences for the plasma membrane: GCVCSSNP (SEQ ID NO: 273), GQTVTTPL(SEQ ID NO: 274), GQELSQHE (SEQ ID NO: 275), GNSPSYNP (SEQ ID NO: 276),GVSGSKGQ (SEQ ID NO: 277), GQTITTPL (SEQ ID NO: 278), GQTLTTPL (SEQ IDNO: 279), GQIFSRSA (SEQ ID NO: 280), GQIHGLSP (SEQ ID NO: 281), GARASVLS(SEQ ID NO: 282), and GCTLSAEE (SEQ ID NO: 283), localisation sequencesfor the endoplasmic reticulum and the nucleus, including GAQVSSQK (SEQID NO: 284), and GAQLSRNT (SEQ ID NO: 285), localisation sequences forthe Golgi apparatus, the nucleus, the cytoplasm and the cytosceleton,including GNAAAAKK (SEQ ID NO: 286), localisation sequences for thecytoplasm and cytosceleton, including GNEASYPL (SEQ ID NO: 287),localisation sequences for the plasma membrane and cytosceleton,including GSSKSKPK (SEQ ID NO: 288), etc. Examples of secretory signalpeptide sequences as defined herein include, without being limitedthereto, signal sequences of classical or non-classical MHC-molecules(e.g. signal sequences of MHC I and II molecules, e.g. of the MHC classI molecule HLA-A*0201), signal sequences of cytokines or immunoglobulinsas defined herein, signal sequences of the invariant chain ofimmunoglobulins or antibodies as defined herein, signal sequences ofLamp1, Tapasin, Erp57, Calreticulin, Calnexin, and further membraneassociated proteins or of proteins associated with the endoplasmicreticulum (ER) or the endosomal-lysosomal compartment. Particularlypreferably, signal sequences of MHC class I molecule HLA-A*0201 may beused according to the present invention. Such an additional componentmay be bound e.g. to a cationic polymer or to any other component of thepolymeric carrier as defined herein. Preferably this signal peptide,localization signal or sequence or nuclear localization signal orsequence (NLS), is bound to the polymeric carrier or to anothercomponent of the polymeric carrier via a (reversible) disulfide bond.For this purpose the (AA) component additionally comprises at least one—SH moiety as defined herein. The binding to any of components of thepolymeric carrier may also be accomplished using an acid-labile bond,preferably via a side chain of any of components of the polymericcarrier, which allows to detach or release the additional component atlower pH-values, e.g. at physiological pH-values as defined herein.

Additionally, according to another alternative, the amino acid component(AA) may be a functional peptide or protein, which may modulate thefunctionality of the polymeric carrier accordingly. Such functionalpeptides or proteins as the amino acid component (AA) preferablycomprise any peptides or proteins as defined herein, e.g. as definedbelow as therapeutically active proteins. According to one alternative,such further functional peptides or proteins may comprise so called cellpenetrating peptides (CPPs) or cationic peptides for transportation.Particularly preferred are CPPs, which induce a pH-mediatedconformational change in the endosome and lead to an improved release ofthe polymeric carrier (in complex with a nucleic acid) from the endosomeby insertion into the lipid layer of the liposome. These cellpenetrating peptides (CPPs) or cationic peptides for transportation, mayinclude, without being limited thereto protamine, nucleoline, spermineor spermidine, oligo- or poly-L-lysine (PLL), basic polypeptides, oligoor poly-arginine, cell penetrating peptides (CPPs), chimeric CPPs, suchas Transportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat(HIV), Tat-derived peptides, members of the penetratin family, e.g.Penetratin, Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, pIsl, etc., antimicrobial-derived CPPs e.g.Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP,MAP, KALA, PpTG20, Loligomere, FGF, Lactoferrin, histones, VP22 derivedor analog peptides, HSV, VP22 (Herpes simplex), MAP, KALA or proteintransduction domains (PTDs, PpT620, prolin-rich peptides, arginine-richpeptides, lysine-rich peptides, Pep-1, L-oligomers, Calcitoninpeptide(s), etc. Such an amino acid component (AA) may also be bound toany component of the polymeric carrier as defined herein. Preferably itis bound to the polymeric carrier or to another component of thepolymeric carrier via a (reversible) disulfide bond. For the abovepurpose, the amino acid component (AA) preferably comprises at least one—SH moiety as defined herein. The binding to any of components of thepolymeric carrier may also be accomplished using an SH-moiety or anacid-labile bond, preferably via a side chain of any of components ofthe polymeric carrier which allows to detach or release the additionalcomponent at lower pH-values, e.g. at physiological pH-values as definedherein.

According to a last alternative, the amino acid component (AA) mayconsist of any peptide or protein which can execute any favorablefunction in the cell. Particularly preferred are peptides or proteinsselected from therapeutically active proteins or peptides, fromantigens, e.g. tumour antigens, pathogenic antigens (animal antigens,viral antigens, protozoal antigens, bacterial antigens, allergicantigens), autoimmune antigens, or further antigens, from allergens,from antibodies, from immunostimulatory proteins or peptides, fromantigen-specific T-cell receptors, or from any other protein or peptidesuitable for a specific (therapeutic) application as defined below forcoding nucleic acids. Particularly preferred are peptide epitopes fromantigens as defined herein.

The polymeric carrier may comprise at least one of the above mentionedcationic or polycationic peptides, proteins or polymers or furthercomponents, e.g. (AA), wherein any of the above alternatives may becombined with each other, and may be formed by polymerizing same in apolymerization condensation reaction via their —SH-moieties.

According to another aspect, the polymeric carrier of the inventivepolymeric carrier cargo complex or single components thereof, e.g. ofthe above mentioned cationic or polycationic peptides, proteins orpolymers or further components, e.g. (AA), may be further modified witha ligand, preferably a carbohydrate, more preferably a sugar, even morepreferably mannose. Preferably this ligand is bound to the polymericcarrier or to a component of the polymeric carrier via a (reversible)disulfide bond or via Michael addition. In the case that the ligand isbound by a disulfide bond the ligand additionally comprises at least one—SH-moiety. These ligands may be used to direct the inventive polymericcarrier cargo complex to specific target cells (e.g. hepatocytes orantigen-presenting cells). In this context mannose is particularpreferred as ligand in the case that dendritic cells are the targetespecially for vaccination or adjuvant purposes.

According to a further aspect of the invention, the inventive polymericcarrier cargo complex may comprise (AA) components as defined abovewhich do not comprise —SH moieties. These (AA) components can be addedbefore or during the complexation reaction of the at least one nucleicacid molecule. Thereby, the (AA) component(s) is/are (non-covalently)incorporated into the inventive polymeric carrier cargo complex withoutinclusion of the (AA) component(s) in the polymeric carrier itself by(covalent) polymerization.

According to one specific embodiment, the entire inventive polymericcarrier cargo complex may be formed by a polymerization condensation (ofat least one) of the above mentioned cationic or polycationic peptides,proteins or polymers or further components, e.g. (AA), via their—SH-moieties in a first step and complexing the nucleic acid to such apolymeric carrier in a second step. The polymeric carrier may thuscontain a number of at least one or even more of the same or differentof the above defined cationic or polycationic peptides, proteins orpolymers or further components, e.g. (AA), the number preferablydetermined by the above range.

According to one alternative specific embodiment, the inventivepolymeric carrier cargo complex is formed by carrying out thepolymerization condensation of at least one of the above mentionedcationic or polycationic peptides, proteins or polymers or furthercomponents, e.g. (AA), via their —SH-moieties simultaneously tocomplexing the nucleic acid cargo to the (in situ prepared) polymericcarrier. Likewise, the polymeric carrier may thus also here contain anumber of at least one or even more of the same or different of theabove defined cationic or polycationic peptides, proteins or polymers orfurther components, e.g. (AA), the number preferably determined by theabove range.

The inventive polymeric carrier cargo complex additionally comprises asa cargo at least one nucleic acid (molecule). In the context of thepresent invention, such a nucleic acid molecule may be any suitablenucleic acid, selected e.g. from any (single-stranded ordouble-stranded) DNA, preferably, without being limited thereto, e.g.genomic DNA, single-stranded DNA molecules, double-stranded DNAmolecules, coding DNA, DNA primers, DNA probes, immunostimulatory DNA, a(short) DNA oligonucleotide ((short) oligodesoxyribonucleotides), or maybe selected e.g. from any PNA (peptide nucleic acid) or may be selectede.g. from any (single-stranded or double-stranded) RNA, preferably,without being limited thereto, a (short) RNA oligonucleotide ((short)oligoribonucleotide), a coding RNA, a messenger RNA (mRNA), animmunostimulatory RNA, a small interfering RNA (siRNA), an antisenseRNA, a micro RNA, a small nuclear RNA (snRNA), a small-hairpin (sh) RNAor riboswitches, ribozymes or aptamers; etc. The nucleic acid moleculeof the inventive polymeric carrier cargo complex may also be a ribosomalRNA (rRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), or a viralRNA (vRNA). Preferably, the nucleic acid molecule of the inventivepolymeric carrier cargo complex is an RNA. More preferably, the nucleicacid molecule of the inventive polymeric carrier cargo complex is a(linear) single-stranded RNA, even more preferably an mRNA or animmunostimulatory RNA. In the context of the present invention, an mRNAis typically an RNA, which is composed of several structural elements,e.g. an optional 5′-CAP structure, an optional 5′-UTR region, anupstream positioned ribosomal binding site followed by a coding region,an optional 3′-UTR region, which may be followed by a poly-A tail(and/or a poly-C-tail). An mRNA may occur as a mono-, di-, or evenmulticistronic RNA, i.e. a RNA which carries the coding sequences ofone, two or more proteins or peptides. Such coding sequences in di-, oreven multicistronic mRNA may be separated by at least one IRES sequence,e.g. as defined herein.

Furthermore, the nucleic acid of the inventive polymeric carrier cargocomplex may be a single- or a double-stranded nucleic acid (molecule)(which may also be regarded as a nucleic acid (molecule) due tonon-covalent association of two single-stranded nucleic acid(s)(molecules)) or a partially double-stranded or partially single strandednucleic acid, which are at least partially self complementary (both ofthese partially double-stranded or partially single stranded nucleicacid molecules are typically formed by a longer and a shortersingle-stranded nucleic acid molecule or by two single stranded nucleicacid molecules, which are about equal in length, wherein onesingle-stranded nucleic acid molecule is in part complementary to theother single-stranded nucleic acid molecule and both thus form adouble-stranded nucleic acid molecule in this region, i.e. a partiallydouble-stranded or partially single stranded nucleic acid (molecule).Preferably, the nucleic acid (molecule) may be a single-stranded nucleicacid molecule. Furthermore, the nucleic acid (molecule) may be acircular or linear nucleic acid molecule, preferably a linear nucleicacid molecule.

According to one alternative, the nucleic acid molecule of the inventivepolymeric carrier cargo complex may be a coding nucleic acid, e.g. a DNAor RNA. Such a coding DNA or RNA may be any DNA or RNA as definedherein. Preferably, such a coding DNA or RNA may be a single- or adouble-stranded DNA or RNA, more preferably a single-stranded DNA orRNA, and/or a circular or linear DNA or RNA, more preferably a linearDNA or RNA. Even more preferably, the coding DNA or RNA may be a(linear) single-stranded DNA or RNA. Most preferably, the nucleic acidmolecule according to the present invention may be a ((linear)single-stranded) messenger RNA (mRNA). Such an mRNA may occur as amono-, di-, or even multicistronic RNA, i.e. an RNA which carries thecoding sequences of one, two or more proteins or peptides. Such codingsequences in di-, or even multicistronic mRNA may be separated by atleast one IRES sequence, e.g. as defined herein.

Coding Nucleic Acids:

The nucleic acid molecule of the inventive polymeric carrier cargocomplex may encode a protein or a peptide, which may be selected,without being restricted thereto, e.g. from therapeutically activeproteins or peptides, including adjuvant proteins, from antigens, e.g.tumour antigens, pathogenic antigens (e.g. selected, from animalantigens, from viral antigens, from protozoal antigens, from bacterialantigens), allergenic antigens, autoimmune antigens, or furtherantigens, from allergens, from antibodies, from immunostimulatoryproteins or peptides, from antigen-specific T-cell receptors, or fromany other protein or peptide suitable for a specific (therapeutic)application, wherein the coding nucleic acid may be transported into acell, a tissue or an organism and the protein may be expressedsubsequently in this cell, tissue or organism.

a) Therapeutically Active Proteins

In the context of the present invention, therapeutically active proteinsor peptides may be encoded by the nucleic acid molecule of the hereindefined inventive polymeric carrier cargo complex. Therapeuticallyactive proteins are defined herein as proteins which have an effect onhealing, prevent prophylactically or treat therapeutically a disease,preferably as defined herein, or are proteins of which an individual isin need of. These may be selected from any naturally or syntheticallydesigned occurring recombinant or isolated protein known to a skilledperson from the prior art. Without being restricted theretotherapeutically active proteins may comprise proteins, capable ofstimulating or inhibiting the signal transduction in the cell, e.g.cytokines, lymphokines, monokines, growth factors, receptors, signaltransduction molecules, transcription factors, etc; anticoagulants;antithrombins; antiallergic proteins; apoptotic factors or apoptosisrelated proteins, therapeutic active enzymes and any protein connectedwith any acquired disease or any hereditary disease.

A therapeutically active protein, which may be encoded by the nucleicacid molecule of the herein defined inventive polymeric carrier cargocomplex, may also be an adjuvant protein. In this context, an adjuvantprotein is preferably to be understood as any protein, which is capableto elicit an innate immune response as defined herein. Preferably, suchan innate immune response comprises activation of a pattern recognitionreceptor, such as e.g. a receptor selected from the Toll-like receptor(TLR) family, including e.g. a Toll like receptor selected from humanTLR1 to TLR10 or from murine Toll like receptors TLR1 to TLR13. Morepreferably, the adjuvant protein is selected from human adjuvantproteins or from pathogenic adjuvant proteins, selected from the groupconsisting of, without being limited thereto, bacterial proteins,protozoan proteins, viral proteins, or fungal proteins, animal proteins,in particular from bacterial adjuvant proteins. In addition, nucleicacids encoding human proteins involved in adjuvant effects (e.g. ligandsof pattern recognition receptors, pattern recoginition receptors,proteins of the signal transduction pathways, transcription factors orcytokines) may be used as well.

b) Antigens

The nucleic acid molecule of the herein defined inventive polymericcarrier cargo complex may alternatively encode an antigen. According tothe present invention, the term “antigen” refers to a substance which isrecognized by the immune system and is capable of triggering anantigen-specific immune response, e.g. by formation of antibodies orantigen-specific T-cells as part of an adaptive immune response. In thiscontext, the first step of an adaptive immune response is the activationof naïve antigen-specific T cells by antigen-presenting cells. Thisoccurs in the lymphoid tissues and organs through which naïve T cellsare constantly passing. The three cell types that can serve asantigen-presenting cells are dendritic cells, macrophages, and B cells.Each of these cells has a distinct function in eliciting immuneresponses. Tissue dendritic cells take up antigens by phagocytosis andmacropinocytosis and are stimulated by infection to migrate to the locallymphoid tissue, where they differentiate into mature dendritic cells.Macrophages ingest particulate antigens such as bacteria and are inducedby infectious agents to express MHC class II molecules. The uniqueability of B cells to bind and internalize soluble protein antigens viatheir receptors may be important to induce T cells. By presenting theantigen on MHC molecules leads to activation of T cells which inducestheir proliferation and differentiation into armed effector T cells. Themost important function of effector T cells is the killing of infectedcells by CD8⁺ cytotoxic T cells and the activation of macrophages by TH1cells which together make up cell-mediated immunity, and the activationof B cells by both TH2 and TH1 cells to produce different classes ofantibody, thus driving the humoral immune response. T cells recognize anantigen by their T cell receptors which does not recognize and bindantigen directly, but instead recognize short peptide fragments e.g. ofpathogens' protein antigens, which are bound to MHC molecules on thesurfaces of other cells.

T cells fall into two major classes that have different effectorfunctions. The two classes are distinguished by the expression of thecell-surface proteins CD4 and CD8. These two types of T cells differ inthe class of MHC molecule that they recognize. There are two classes ofMHC molecules—MHC class I and MHC class II molecules—which differ intheir structure and expression pattern on tissues of the body. CD4⁺ Tcells bind to a MHC class II molecule and CD8⁺ T cells to a MHC class Imolecule. MHC class I and MHC class II molecules have distinctdistributions among cells that reflect the different effector functionsof the T cells that recognize them. MHC class I molecules presentpeptides from pathogens, commonly viruses to CD8⁺ T cells, whichdifferentiate into cytotoxic T cells that are specialized to kill anycell that they specifically recognize. Almost all cells express MHCclass I molecules, although the level of constitutive expression variesfrom one cell type to the next. But not only pathogenic peptides fromviruses are presented by MHC class I molecules, also self-antigens liketumour antigens are presented by them. MHC class I molecules bindpeptides from proteins degraded in the cytosol and transported in theendoplasmic reticulum. Thereby MHC class I molecules on the surface ofcells infected with viruses or other cytosolic pathogens displaypeptides from these pathogen. The CD8⁺ T cells that recognize MHC classI:peptide complexes are specialized to kill any cells displaying foreignpeptides and so rid the body of cells infected with viruses and othercytosolic pathogens. The main function of CD4⁺ T cells (CD4⁺ helper Tcells) that recognize MHC class II molecules is to activate othereffector cells of the immune system. Thus MHC class II molecules arenormally found on B lymphocytes, dendritic cells, and macrophages, cellsthat participate in immune responses, but not on other tissue cells.Macrophages, for example, are activated to kill the intravesicularpathogens they harbour, and B cells to secrete immunoglobulins againstforeign molecules. MHC class II molecules are prevented from binding topeptides in the endoplasmic reticulum and thus MHC class II moleculesbind peptides from proteins which are degraded in endosomes. They cancapture peptides from pathogens that have entered the vesicular systemof macrophages, or from antigens internalized by immature dendriticcells or the immunoglobulin receptors of B cells. Pathogens thataccumulate in large numbers inside macrophage and dendritic cellvesicles tend to stimulate the differentiation of TH1 cells, whereasextracellular antigens tend to stimulate the production of TH2 cells.TH1 cells activate the microbicidal properties of macrophages and induceB cells to make IgG antibodies that are very effective of opsonisingextracellular pathogens for ingestion by phagocytic cells, whereas TH2cells initiate the humoral response by activating naïve B cells tosecrete IgM, and induce the production of weakly opsonising antibodiessuch as IgG1 and IgG3 (mouse) and IgG2 and IgG4 (human) as well as IgAand IgE (mouse and human).

In the context of the present invention, antigens as encoded by thenucleic acid molecule of the herein defined inventive polymeric carriercargo complex typically comprise any antigen, antigenic epitope orantigenic peptide, falling under the above definition, more preferablyprotein and peptide antigens, e.g. tumour antigens, allergenic antigens,auto-immune self-antigens, pathogenic antigens, etc. In particularantigens as encoded by the nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex may be antigens generatedoutside the cell, more typically antigens not derived from the hostorganism (e.g. a human) itself (i.e. non-self antigens) but ratherderived from host cells outside the host organism, e.g. viral antigens,bacterial antigens, fungal antigens, protozoological antigens, animalantigens, allergenic antigens, etc. Allergenic antigens (allergyantigens) are typically antigens, which cause an allergy in a human andmay be derived from either a human or other sources. Additionally,antigens as encoded by the nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex may be furthermore antigensgenerated inside the cell, the tissue or the body. Such antigens includeantigens derived from the host organism (e.g. a human) itself, e.g.tumour antigens, self-antigens or auto-antigens, such as auto-immuneself-antigens, etc., but also (non-self) antigens as defined herein,which have been originally been derived from host cells outside the hostorganism, but which are fragmented or degraded inside the body, tissueor cell, e.g. by (protease) degradation, metabolism, etc.

One class of antigens as encoded by the nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex comprisestumour antigens. “Tumour antigens” are preferably located on the surfaceof the (tumour) cell. Tumour antigens may also be selected fromproteins, which are overexpressed in tumour cells compared to a normalcell. Furthermore, tumour antigens also include antigens expressed incells which are (were) not themselves (or originally not themselves)degenerated but are associated with the supposed tumour. Antigens whichare connected with tumour-supplying vessels or (re)formation thereof, inparticular those antigens which are associated with neovascularization,e.g. growth factors, such as VEGF, bFGF etc., are also included herein.Antigens connected with a tumour furthermore include antigens from cellsor tissues, typically embedding the tumour. Further, some substances(usually proteins or peptides) are expressed in patients suffering(knowingly or not-knowingly) from a cancer disease and they occur inincreased concentrations in the body fluids of said patients. Thesesubstances are also referred to as “tumour antigens”, however they arenot antigens in the stringent meaning of an immune response inducingsubstance. The class of tumour antigens can be divided further intotumour-specific antigens (TSAs) and tumour-associated-antigens (TAAs).TSAs can only be presented by tumour cells and never by normal “healthy”cells. They typically result from a tumour specific mutation. TAAs,which are more common, are usually presented by both tumour and healthycells. These antigens are recognized and the antigen-presenting cell canbe destroyed by cytotoxic T cells. Additionally, tumour antigens canalso occur on the surface of the tumour in the form of, e.g., a mutatedreceptor. In this case, they can be recognized by antibodies. Particularpreferred tumour antigens are selected from the group consisting of 5T4,707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin,alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha-methylacyl-coenzyme Aracemase, ART-4, ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, beta-catenin/m,BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125,calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20,CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m,CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m, coactosin-like protein,collage XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B,CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam,EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1,GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V,gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL,HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE, homeobox NKX3.1,HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT,iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor,kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m, KK-LC-1,K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6,MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4,MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2,MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1,MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrix protein22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen,MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m,NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m,NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP,OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minorbcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PART-1, PATE, PDEF,Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein,proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m,RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC,SIRT2/m, Sp17, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin,survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1,TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b,TRP/INT2, TRP-p8, tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, and WT1. Suchtumour antigens preferably may be selected from the group consisting ofMAGE-A1 (e.g. MAGE-A1 according to accession number M77481), MAGE-A2,MAGE-A3, MAGE-A6 (e.g. MAGE-A6 according to accession number NM_005363),MAGE-C1, MAGE-C2, melan-A (e.g. melan-A according to accession numberNM_005511), GP100 (e.g. GP100 according to accession number M77348),tyrosinase (e.g. tyrosinase according to accession number NM_000372),surviving (e.g. survivin according to accession number AF077350), CEA(e.g. CEA according to accession number NM_004363), Her-2/neu (e.g.Her-2/neu according to accession number M11730), WT1 (e.g. WT1 accordingto accession number NM_000378), PRAME (e.g. PRAME according to accessionnumber NM_006115), EGFRI (epidermal growth factor receptor 1) (e.g.EGFRI (epidermal growth factor receptor 1) according to accession numberAF288738), MUC1, mucin-1 (e.g. mucin-1 according to accession numberNM_002456), SEC61G (e.g. SEC61G according to accession numberNM_014302), hTERT (e.g. hTERT accession number NM_198253), 5T4 (e.g. 5T4according to accession number NM_006670), NY-Eso-1 (e.g. NY-Eso1according to accession number NM_001327), TRP-2 (e.g. TRP-2 according toaccession number NM_001922), STEAP, PCA, PSA, PSMA, etc.

According to another alternative, one further class of antigens asencoded by the nucleic acid molecule of the herein defined inventivepolymeric carrier cargo complex comprises allergenic antigens. Suchallergenic antigens may be selected from antigens derived from differentsources, e.g. from animals, plants, fungi, bacteria, etc. Allergens inthis context include e.g. grasses, pollens, molds, drugs, or numerousenvironmental triggers, etc. Allergenic antigens typically belong todifferent classes of compounds, such as nucleic acids and theirfragments, proteins or peptides and their fragments, carbohydrates,polysaccharides, sugars, lipids, phospholipids, etc. Of particularinterest in the context of the present invention are antigens, which maybe encoded by the nucleic acid molecule of the inventive polymericcarrier cargo complex, i.e. protein or peptide antigens and theirfragments or epitopes, or nucleic acids and their fragments,particularly nucleic acids and their fragments, encoding such protein orpeptide antigens and their fragments or epitopes.

c) Antibodies

According to a further alternative, the nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex may encode anantibody or an antibody fragment. According to the present invention,such an antibody may be selected from any antibody, e.g. anyrecombinantly produced or naturally occurring antibodies, known in theart, in particular antibodies suitable for therapeutic, diagnostic orscientific purposes, or antibodies which have been identified inrelation to specific cancer diseases. Herein, the term “antibody” isused in its broadest sense and specifically covers monoclonal andpolyclonal antibodies (including agonist, antagonist, and blocking orneutralizing antibodies) and antibody species with polyepitopicspecificity. According to the invention, the term “antibody” typicallycomprises any antibody known in the art (e.g. IgM, IgD, IgG, IgA and IgEantibodies), such as naturally occurring antibodies, antibodiesgenerated by immunization in a host organism, antibodies which wereisolated and identified from naturally occurring antibodies orantibodies generated by immunization in a host organism andrecombinantly produced by biomolecular methods known in the art, as wellas chimeric antibodies, human antibodies, humanized antibodies,bispecific antibodies, intrabodies, i.e. antibodies expressed in cellsand optionally localized in specific cell compartments, and fragmentsand variants of the aforementioned antibodies. In general, an antibodyconsists of a light chain and a heavy chain both having variable andconstant domains. The light chain consists of an N-terminal variabledomain, V_(L), and a C-terminal constant domain, C_(L). In contrast, theheavy chain of the IgG antibody, for example, is comprised of anN-terminal variable domain, V_(H), and three constant domains, C_(H)1,C_(H)2 and C_(H)3.

In the context of the present invention, antibodies as encoded by thenucleic acid molecule of the herein defined inventive polymeric carriercargo complex may preferably comprise full-length antibodies, i.e.antibodies composed of the full heavy and full light chains, asdescribed above. However, derivatives of antibodies such as antibodyfragments, variants or adducts may also be encoded by the nucleic acidmolecule of the herein defined inventive inventive polymeric carriercargo complex. Antibody fragments are preferably selected from Fab,Fab′, F(ab′)₂, Fc, Facb, pFc′, Fd and Fv fragments of the aforementioned(full-length) antibodies. In general, antibody fragments are known inthe art. For example, a Fab (“fragment, antigen binding”) fragment iscomposed of one constant and one variable domain of each of the heavyand the light chain. The two variable domains bind the epitope onspecific antigens. The two chains are connected via a disulfide linkage.A scFv (“single chain variable fragment”) fragment, for example,typically consists of the variable domains of the light and heavychains. The domains are linked by an artificial linkage, in general apolypeptide linkage such as a peptide composed of 15-25 glycine, prolineand/or serine residues.

In the present context it is preferable that the different chains of theantibody or antibody fragment are encoded by a multicistronic nucleicacid molecule. Alternatively, the different strains of the antibody orantibody fragment are encoded by several monocistronic nucleic acid(s)(sequences).

siRNA:

According to a further alternative, the nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex may be in theform of dsRNA, preferably siRNA. A dsRNA, or a siRNA, is of interestparticularly in connection with the phenomenon of RNA interference. Thein vitro technique of RNA interference (RNAi) is based ondouble-stranded RNA molecules (dsRNA), which trigger thesequence-specific suppression of gene expression (Zamore (2001) Nat.Struct. Biol. 9: 746-750; Sharp (2001) Genes Dev. 5:485-490: Hannon(2002) Nature 41: 244-251). In the transfection of mammalian cells withlong dsRNA, the activation of protein kinase R and RnaseL brings aboutunspecific effects, such as, for example, an interferon response (Starket al. (1998) Annu. Rev. Biochem. 67: 227-264; He and Katze (2002) ViralImmunol. 15: 95-119). These unspecific effects are avoided when shorter,for example 21- to 23-mer, so-called siRNA (small interfering RNA), isused, because unspecific effects are not triggered by siRNA that isshorter than 30 bp (Elbashir et al. (2001) Nature 411: 494-498).

The nucleic acid molecule of the herein inventive polymeric carriercargo complex may thus be a double-stranded RNA (dsRNA) having a lengthof from 17 to 29, preferably from 19 to 25, and preferably is at least90%, more preferably 95% and especially 100% (of the nucleotides of adsRNA) complementary to a section of the nucleic acid molecule of a(therapeutically relevant) protein or antigen described (as activeingredient) hereinbefore or of any further protein as described herein,either a coding or a non-coding section, preferably a coding section.Such a (section of the) nucleic acid molecule may be termed herein a“target sequence” and may be any nucleic acid molecule as definedherein, preferably a genomic DNA, a cDNA, a RNA, e.g. an mRNA, etc. 90%complementary means that with a length of a dsRNA described herein of,for example, 20 nucleotides, the dsRNA contains not more than 2nucleotides showing no complementarity with the corresponding section ofthe target sequence. The sequence of the double-stranded RNA usedaccording to the invention is, however, preferably wholly complementaryin its general structure with a section of the target sequence. In thiscontext the nucleic acid molecule of the inventive polymeric carriercargo complex may be a dsRNA having the general structure5′-(N17-29)-3′, preferably having the general structure 5′-(N19-25)-3′,more preferably having the general structure 5′-(N19-24)-3′, or yet morepreferably having the general structure 5′-(N21-23)-3′, wherein for eachgeneral structure each N is a (preferably different) nucleotide of asection of the target sequence, preferably being selected from acontinuous number of 17 to 29 nucleotides of a section of the targetsequence, and being present in the general structure 5′-(N17-29)-3′ intheir natural order. In principle, all the sections having a length offrom 17 to 29, preferably from 19 to 25, base pairs that occur in thetarget sequence can serve for preparation of a dsRNA as defined herein.Equally, dsRNAs used as nucleic acid molecule of the inventive polymericcarrier cargo complex can also be directed against nucleotide sequencesof a (therapeutically relevant) protein or antigen described (as activeingredient) hereinbefore that do not lie in the coding region, inparticular in the 5′ non-coding region of the target sequence, forexample, therefore, against non-coding regions of the target sequencehaving a regulatory function. The target sequence of the dsRNA used asnucleic acid molecule of the inventive polymeric carrier cargo complexcan therefore lie in the translated and untranslated region of thetarget sequence and/or in the region of the control elements of aprotein or antigen described hereinbefore. The target sequence for adsRNA used as the nucleic acid molecule of the inventive polymericcarrier cargo complex can also lie in the overlapping region ofuntranslated and translated sequence; in particular, the target sequencecan comprise at least one nucleotide upstream of the start triplet ofthe coding region, e.g. of a genomic DNA, a cDNA, a RNA, or an mRNA,etc.

Immunostimulatory Nucleic Acids:

a) Immunostimulatory CpG Nucleic Acids:

According to another alternative, the nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex may be in theform of a(n) (immunostimulatory) CpG nucleic acid, in particular CpG-RNAor CpG-DNA, which preferably induces an innate immune response. ACpG-RNA or CpG-DNA used according to the invention can be asingle-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA),a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (dsCpG-RNA). The CpG nucleic acid used according to the invention ispreferably in the form of CpG-RNA, more preferably in the form ofsingle-stranded CpG-RNA (ss CpG-RNA). Also preferably, such CpG nucleicacids have a length as described above. Preferably the CpG motifs areunmethylated.

b) Immunostimulatory RNA (isRNA):

Likewise, according to a further alternative, the (immunostimulatory)nucleic acid molecule of the inventive polymeric carrier cargo complexmay be in the form of an immunostimulatory RNA (isRNA), which preferablyelicits an innate immune response. Such an immunostimulatory RNA may beany (double-stranded or single-stranded) RNA, e.g. a coding RNA, asdefined herein. Preferably, the immunostimulatory RNA may be asingle-stranded, a double-stranded or a partially double-stranded RNA,more preferably a single-stranded RNA, and/or a circular or linear RNA,more preferably a linear RNA. More preferably, the immunostimulatory RNAmay be a (linear) single-stranded RNA. Even more preferably, theimmunostimulatory RNA may be a (long) (linear) single-stranded)non-coding RNA. In this context it is particular preferred that theisRNA carries a triphosphate at its 5′-end which is the case for invitro transcribed RNA. An immunostimulatory RNA may also occur as ashort RNA oligonucleotide as defined herein. An immunostimulatory RNA asused herein may furthermore be selected from any class of RNA molecules,found in nature or being prepared synthetically, and which can induce aninnate immune response and may support an adaptive immune responseinduced by an antigen. In this context, an immune response may occur invarious ways. A substantial factor for a suitable (adaptive) immuneresponse is the stimulation of different T-cell sub-populations.T-lymphocytes are typically divided into two sub-populations, theT-helper 1 (Th1) cells and the T-helper 2 (Th2) cells, with which theimmune system is capable of destroying intracellular (Th1) andextracellular (Th2) pathogens (e.g. antigens). The two Th cellpopulations differ in the pattern of the effector proteins (cytokines)produced by them. Thus, Th1 cells assist the cellular immune response byactivation of macrophages and cytotoxic T-cells. Th2 cells, on the otherhand, promote the humoral immune response by stimulation of B-cells forconversion into plasma cells and by formation of antibodies (e.g.against antigens). The Th1/Th2 ratio is therefore of great importance inthe induction and maintenance of an adaptive immune response. Inconnection with the present invention, the Th1/Th2 ratio of the(adaptive) immune response is preferably shifted in the directiontowards the cellular response (Th1 response) and a cellular immuneresponse is thereby induced. According to one example, the innate immunesystem which may support an adaptive immune response, may be activatedby ligands of Toll-like receptors (TLRs). TLRs are a family of highlyconserved pattern recognition receptor (PRR) polypeptides that recognizepathogen-associated molecular patterns (PAMPs) and play a critical rolein innate immunity in mammals. Currently at least thirteen familymembers, designated TLR1-TLR13 (Toll-like receptors: TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13), havebeen identified. Furthermore, a number of specific TLR ligands have beenidentified. It was e.g. found that unmethylated bacterial DNA andsynthetic analogs thereof (CpG DNA) are ligands for TLR9 (Hemmi H et al.(2000) Nature 408:740-5; Bauer S et al. (2001) Proc NatlAcadSci USA 98,9237-42). Furthermore, it has been reported that ligands for certainTLRs include certain nucleic acid molecules and that certain types ofRNA are immunostimulatory in a sequence-independent orsequence-dependent manner, wherein these various immunostimulatory RNAsmay e.g. stimulate TLR3, TLR7, or TLR8, or intracellular receptors suchas RIG-I, MDA-5, etc. E.g. Lipford et al. determined certainG,U-containing oligoribonucleotides as immunostimulatory by acting viaTLR7 and TLR8 (see WO 03/086280). The immunostimulatory G,U-containingoligoribonucleotides described by Lipford et al. were believed to bederivable from RNA sources including ribosomal RNA, transfer RNA,messenger RNA, and viral RNA.

The immunostimulatory RNA (isRNA) used as the nucleic acid molecule ofthe herein defined inventive polymeric carrier cargo complex may thuscomprise any RNA sequence known to be immunostimulatory, including,without being limited thereto, RNA sequences representing and/orencoding ligands of TLRs, preferably selected from human family membersTLR1-TLR10 or murine family members TLR1-TLR13, more preferably selectedfrom (human) family members TLR1-TLR10, even more preferably from TLR7and TLR8, ligands for intracellular receptors for RNA (such as RIG-I orMDA-5, etc.) (see e.g. Meylan, E., Tschopp, J. (2006). Toll-likereceptors and RNA helicases: two parallel ways to trigger antiviralresponses. Mol. Cell 22, 561-569), or any other immunostimulatory RNAsequence. Furthermore, (classes of) immunostimulatory RNA molecules,used as the nucleic acid molecule of the inventive polymeric carriercargo complex may include any other RNA capable of eliciting an innateimmune response. Without being limited thereto, such animmunostimulatory RNA may include ribosomal RNA (rRNA), transfer RNA(tRNA), messenger RNA (mRNA), and viral RNA (vRNA). Such animmunostimulatory RNA may comprise a length of 1000 to 5000, of 500 to5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of5 to 50 or of 5 to 30 nucleotides.

According to a particularly preferred embodiment, such immunostimulatorynucleic acid sequences is preferably RNA preferably consisting of orcomprising a nucleic acid of formula (II) or (III):

G_(l)X_(m)G_(n),  (formula (II))

wherein:

-   -   G is guanosine, uracil or an analogue of guanosine or uracil;    -   X is guanosine, uracil, adenosine, thymidine, cytosine or an        analogue of the above-mentioned nucleotides;    -   l is an integer from 1 to 40,        -   wherein        -   when l=1 G is guanosine or an analogue thereof,        -   when l>1 at least 50% of the nucleotides are guanosine or an            analogue thereof;    -   m is an integer and is at least 3;        -   wherein        -   when m=3 X is uracil or an analogue thereof,        -   when m>3 at least 3 successive uracils or analogues of            uracil occur;    -   n is an integer from 1 to 40,        -   wherein        -   when n=1 G is guanosine or an analogue thereof,        -   when n>1 at least 50% of the nucleotides are guanosine or an            analogue thereof.

C_(l)X_(m)C_(n),  (formula (III))

wherein:

-   -   C is cytosine, uracil or an analogue of cytosine or uracil;    -   X is guanosine, uracil, adenosine, thymidine, cytosine or an        analogue of the above-mentioned nucleotides;    -   l is an integer from 1 to 40,        -   wherein        -   when l=1 C is cytosine or an analogue thereof,        -   when l>1 at least 50% of the nucleotides are cytosine or an            analogue thereof;    -   m is an integer and is at least 3;        -   wherein        -   when m=3 X is uracil or an analogue thereof,        -   when m>3 at least 3 successive uracils or analogues of            uracil occur;    -   n is an integer from 1 to 40,        -   wherein        -   when n=1 C is cytosine or an analogue thereof,        -   when n>1 at least 50% of the nucleotides are cytosine or an            analogue thereof.

The nucleic acids of formula (II) or (III), which may be used thenucleic acid cargo of the inventive polymeric carrier cargo complex maybe relatively short nucleic acid molecules with a typical length ofapproximately from 5 to 100 (but may also be longer than 100 nucleotidesfor specific embodiments, e.g. up to 200 nucleotides), from 5 to 90 orfrom 5 to 80 nucleotides, preferably a length of approximately from 5 to70, more preferably a length of approximately from 8 to 60 and, morepreferably a length of approximately from 15 to 60 nucleotides, morepreferably from 20 to 60, most preferably from 30 to 60 nucleotides. Ifthe nucleic acid of the inventive nucleic acid cargo complex has amaximum length of e.g. 100 nucleotides, m will typically be <=98. Thenumber of nucleotides G in the nucleic acid of formula (II) isdetermined by l or n. l and n, independently of one another, are each aninteger from 1 to 40, wherein when l or n=1 G is guanosine or ananalogue thereof, and when l or n>1 at least 50% of the nucleotides areguanosine or an analogue thereof. For example, without implying anylimitation, when l or n=4 G_(l) or G_(n) can be, for example, a GUGU,GGUU, UGUG, UUGG, GUUG, GGGU, GGUG, GUGG, UGGG or GGGG, etc.; when l orn=5 G_(l) or G_(n) can be, for example, a GGGUU, GGUGU, GUGGU, UGGGU,UGGUG, UGUGG, UUGGG, GUGUG, GGGGU, GGGUG, GGUGG, GUGGG, UGGGG, or GGGGG,etc.; etc. A nucleotide adjacent to X_(m) in the nucleic acid of formula(II) according to the invention is preferably not a uracil. Similarly,the number of nucleotides C in the nucleic acid of formula (III)according to the invention is determined by l or n. l and n,independently of one another, are each an integer from 1 to 40, whereinwhen l or n=1 C is cytosine or an analogue thereof, and when l or n>1 atleast 50% of the nucleotides are cytosine or an analogue thereof. Forexample, without implying any limitation, when l or n=4, C_(l) or C_(n)can be, for example, a CUCU, CCUU, UCUC, UUCC, CUUC, CCCU, CCUC, CUCC,UCCC or CCCC, etc.; when l or n=5 C_(l) or C_(n) can be, for example, aCCCUU, CCUCU, CUCCU, UCCCU, UCCUC, UCUCC, UUCCC, CUCUC, CCCCU, CCCUC,CCUCC, CUCCC, UCCCC, or CCCCC, etc.; etc. A nucleotide adjacent to X_(m)in the nucleic acid of formula (III) according to the invention ispreferably not a uracil. Preferably, for formula (II), when 1 or n>1, atleast 60%, 70%, 80%, 90% or even 100% of the nucleotides are guanosineor an analogue thereof, as defined above. The remaining nucleotides to100% (when guanosine constitutes less than 100% of the nucleotides) inthe flanking sequences G_(l) and/or G_(n) are uracil or an analoguethereof, as defined hereinbefore. Also preferably, 1 and n,independently of one another, are each an integer from 2 to 30, morepreferably an integer from 2 to 20 and yet more preferably an integerfrom 2 to 15. The lower limit of 1 or n can be varied if necessary andis at least 1, preferably at least 2, more preferably at least 3, 4, 5,6, 7, 8, 9 or 10. This definition applies correspondingly to formula(III).

According to a particularly preferred embodiment, a nucleic acidaccording to any of formulas (II) or (III) above, which may be used asnucleic acid of the inventive polymeric carrier cargo complex, may beselected from a sequence consisting or comprising any of the followingsequences:

(SEQ ID NO: 289) GGUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 290)GGGGGUUUUUUUUUUGGGGG; (SEQ ID NO: 291)GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 292)GUGUGUGUGUGUUUUUUUUUUUUUUUUGUGUGUGUGUGU; (SEQ ID NO: 293)GGUUGGUUGGUUUUUUUUUUUUUUUUUGGUUGGUUGGUU; (SEQ ID NO: 294)GGGGGGGGGUUUGGGGGGGG; (SEQ ID NO: 295) GGGGGGGGUUUUGGGGGGGG;(SEQ ID NO: 296) GGGGGGGUUUUUUGGGGGGG; (SEQ ID NO: 297)GGGGGGGUUUUUUUGGGGGG; (SEQ ID NO: 298) GGGGGGUUUUUUUUGGGGGG;(SEQ ID NO: 299) GGGGGGUUUUUUUUUGGGGG; (SEQ ID NO: 300)GGGGGGUUUUUUUUUUGGGG; (SEQ ID NO: 301) GGGGGUUUUUUUUUUUGGGG;(SEQ ID NO: 302) GGGGGUUUUUUUUUUUUGGG; (SEQ ID NO: 303)GGGGUUUUUUUUUUUUUGGG; (SEQ ID NO: 304) GGGGUUUUUUUUUUUUUUGG;(SEQ ID NO: 305) GGUUUUUUUUUUUUUUUUGG; (SEQ ID NO: 306)GUUUUUUUUUUUUUUUUUUG; (SEQ ID NO: 307) GGGGGGGGGGUUUGGGGGGGGG;(SEQ ID NO: 308) GGGGGGGGGUUUUGGGGGGGGG; (SEQ ID NO: 309)GGGGGGGGUUUUUUGGGGGGGG; (SEQ ID NO: 310) GGGGGGGGUUUUUUUGGGGGGG;(SEQ ID NO: 311) GGGGGGGUUUUUUUUGGGGGGG; (SEQ ID NO: 312)GGGGGGGUUUUUUUUUGGGGGG; (SEQ ID NO: 313) GGGGGGGUUUUUUUUUUGGGGG;(SEQ ID NO: 314) GGGGGGUUUUUUUUUUUGGGGG; (SEQ ID NO: 315)GGGGGGUUUUUUUUUUUUGGGG; (SEQ ID NO: 316) GGGGGUUUUUUUUUUUUUGGGG;(SEQ ID NO: 317) GGGGGUUUUUUUUUUUUUUGGG; (SEQ ID NO: 318)GGGUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 319) GGUUUUUUUUUUUUUUUUUUGG;(SEQ ID NO: 320) GGGGGGGGGGGUUUGGGGGGGGGG; (SEQ ID NO: 321)GGGGGGGGGGUUUUGGGGGGGGGG; (SEQ ID NO: 322) GGGGGGGGGUUUUUUGGGGGGGGG;(SEQ ID NO: 323) GGGGGGGGUUUUUUUUGGGGGGGG; (SEQ ID NO: 324)GGGGGGGGUUUUUUUUUGGGGGGG; (SEQ ID NO: 325) GGGGGGGGGUUUUUUUGGGGGGGG;(SEQ ID NO: 326) GGGGGGGGUUUUUUUUUUGGGGGG; (SEQ ID NO: 327)GGGGGGGUUUUUUUUUUUGGGGGG; (SEQ ID NO: 328) GGGGGGGUUUUUUUUUUUUGGGGG;(SEQ ID NO: 329) GGGGGGUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 330)GGGGGGUUUUUUUUUUUUUUGGGG; (SEQ ID NO: 331) GGGGUUUUUUUUUUUUUUUUGGGG;(SEQ ID NO: 332) GGGUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 333)GUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUG; (SEQ ID NO: 334)GGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGG; (SEQ ID NO: 335)GGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 336)GGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGG; (SEQ ID NO: 337)GGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGG; (SEQ ID NO: 338)GGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGG; (SEQ ID NO: 339)GGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGG; (SEQ ID NO: 340)GGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGG; (SEQ ID NO: 341)GGGGGGGGGUUUUUUUUUUUUUUUUUUUUUUUUUUUUUUGGGGGGGG; (SEQ ID NO: 342)GGUUUGG; (SEQ ID NO: 343) GGUUUUGG; (SEQ ID NO: 344) GGUUUUUGG;(SEQ ID NO: 345) GGUUUUUUGG; (SEQ ID NO: 346) GGUUUUUUUGG;(SEQ ID NO: 347) GGUUUUUUUUGG; (SEQ ID NO: 348) GGUUUUUUUUUGG;(SEQ ID NO: 349) GGUUUUUUUUUUGG; (SEQ ID NO: 350) GGUUUUUUUUUUUGG;(SEQ ID NO: 351) GGUUUUUUUUUUUUGG; (SEQ ID NO: 352) GGUUUUUUUUUUUUUGG;(SEQ ID NO: 353) GGUUUUUUUUUUUUUUGG; (SEQ ID NO: 354GGUUUUUUUUUUUUUUUGG); (SEQ ID NO: 355) GGGUUUGGG; (SEQ ID NO: 356)GGGUUUUGGG; (SEQ ID NO: 357) GGGUUUUUGGG; (SEQ ID NO: 358) GGGUUUUUUGGG;(SEQ ID NO: 359) GGGUUUUUUUGGG; (SEQ ID NO: 360) GGGUUUUUUUUGGG;(SEQ ID NO: 361) GGGUUUUUUUUUGGG; (SEQ ID NO: 362) GGGUUUUUUUUUUGGG;(SEQ ID NO: 363) GGGUUUUUUUUUUUGGG; (SEQ ID NO: 364) GGGUUUUUUUUUUUUGGG;(SEQ ID NO: 365) GGGUUUUUUUUUUUUUGGG; (SEQ ID NO: 366)GGGUUUUUUUUUUUUUUUGGGUUUUUUUUUUUUUUUGGGUUUUUUUUUUUU UUUGGG;(SEQ ID NO: 367) GGGUUUUUUUUUUUUUUUGGGGGGUUUUUUUUUUUUUUUGGG;(SEQ ID NO: 368) GGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUGGGUUUG GG;(short GU-rich, SEQ ID NO: 369) GGUUUUUUUUUUUUUUUGGG or (SEQ ID NO: 370)CCCUUUUUUUUUUUUUUUCCCUUUUUUUUUUUUUUUCCCUUUUUUUUUUUU UUUCCC(SEQ ID NO: 371) CCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCCUUUCCC(SEQ ID NO: 372) CCCUUUUUUUUUUUUUUUCCCCCCUUUUUUUUUUUUUUUCCC

or from a sequence having at least 60%, 70%, 80%, 90%, or even 95%sequence identity with any of these sequences

According to a further particularly preferred embodiment, suchimmunostimulatory nucleic acid sequences particularly isRNA consist ofor comprise a nucleic acid of formula (IV) or (V):

(N_(u)G_(l)X_(n)G_(n)N_(v))_(a),  (formula (IV))

wherein:

-   -   G is guanosine (guanine), uridine (uracil) or an analogue of        guanosine (guanine) or uridine (uracil), preferably guanosine        (guanine) or an analogue thereof;    -   X is guanosine (guanine), uridine (uracil), adenosine (adenine),        thymidine (thymine), cytidine (cytosine), or an analogue of        these nucleotides (nucleosides), preferably uridine (uracil) or        an analogue thereof;    -   N is a nucleic acid sequence having a length of about 4 to 50,        preferably of about 4 to 40, more preferably of about 4 to 30 or        4 to 20 nucleic acids, each N independently being selected from        guanosine (guanine), uridine (uracil), adenosine (adenine),        thymidine (thymine), cytidine (cytosine) or an analogue of these        nucleotides (nucleosides);    -   a is an integer from 1 to 20, preferably from 1 to 15, most        preferably from 1 to 10;    -   l is an integer from 1 to 40,        -   wherein when l=1, G is guanosine (guanine) or an analogue            thereof,            -   when l>1, at least 50% of these nucleotides                (nucleosides) are guanosine (guanine) or an analogue                thereof;    -   m is an integer and is at least 3;        -   wherein when m=3, X is uridine (uracil) or an analogue            thereof, and            -   when m>3, at least 3 successive uridines (uracils) or                analogues of uridine (uracil) occur;    -   n is an integer from 1 to 40,        -   wherein when n=1, G is guanosine (guanine) or an analogue            thereof,            -   when n>1, at least 50% of these nucleotides                (nucleosides) are guanosine (guanine) or an analogue                thereof;    -   u,v may be independently from each other an integer from 0 to        50,        -   preferably wherein when u=0, v≥1, or            -   when v=0, u≥1;

wherein the nucleic acid molecule of formula (IV) has a length of atleast 50 nucleotides, preferably of at least 100 nucleotides, morepreferably of at least 150 nucleotides, even more preferably of at least200 nucleotides and most preferably of at least 250 nucleotides.

(N_(u)C_(l)X_(m)C_(n)N_(v))_(a),  (formula (V))

wherein:

-   -   C is cytidine (cytosine), uridine (uracil) or an analogue of        cytidine (cytosine) or uridine (uracil), preferably cytidine        (cytosine) or an analogue thereof;    -   X is guanosine (guanine), uridine (uracil), adenosine (adenine),        thymidine (thymine), cytidine (cytosine) or an analogue of the        above-mentioned nucleotides (nucleosides), preferably uridine        (uracil) or an analogue thereof;    -   N is each a nucleic acid sequence having independent from each        other a length of about 4 to 50, preferably of about 4 to 40,        more preferably of about 4 to 30 or 4 to 20 nucleic acids, each        N independently being selected from guanosine (guanine), uridine        (uracil), adenosine (adenine), thymidine (thymine), cytidine        (cytosine) or an analogue of these nucleotides (nucleosides);    -   a is an integer from 1 to 20, preferably from 1 to 15, most        preferably from 1 to 10;    -   l is an integer from 1 to 40,        -   wherein when l=1, C is cytidine (cytosine) or an analogue            thereof,            -   when l>1, at least 50% of these nucleotides                (nucleosides) are cytidine (cytosine) or an analogue            -   thereof;    -   m is an integer and is at least 3;        -   wherein when m=3, X is uridine (uracil) or an analogue            thereof,            -   when m>3, at least 3 successive uridines (uracils) or                analogues of uridine (uracil) occur;    -   n is an integer from 1 to 40,        -   wherein when n=1, C is cytidine (cytosine) or an analogue            thereof,            -   when n>1, at least 50% of these nucleotides                (nucleosides) are cytidine (cytosine) or an analogue            -   thereof.    -   u, v may be independently from each other an integer from 0 to        50,        -   preferably wherein when u=0, v≥1, or            -   when v=0, u≥1;

wherein the nucleic acid molecule of formula (V) according to theinvention has a length of at least 50 nucleotides, preferably of atleast 100 nucleotides, more preferably of at least 150 nucleotides, evenmore preferably of at least 200 nucleotides and most preferably of atleast 250 nucleotides.

For formula (V), any of the definitions given above for elements N (i.e.N_(u) and N_(v)) and X (X_(m)), particularly the core structure asdefined above, as well as for integers a, l, m, n, u and v, similarlyapply to elements of formula (V) correspondingly, wherein in formula (V)the core structure is defined by C_(l)X_(m)C_(n). The definition ofbordering elements N_(u) and N_(v) is identical to the definitions givenabove for N_(u) and N_(v).

According to a very particularly preferred embodiment, the inventivenucleic acid molecule according to formula (IV) may be selected frome.g. any of the following sequences:

(SEQ ID NO: 373) UAGCGAAGCUCUUGGACCUAGGUUUUUUUUUUUUUUUGGGUGCGUUCCUAGAAGUACACG (SEQ ID NO: 374)UAGCGAAGCUCUUGGACCUAGGUUUUUUUUUUUUUUUGGGUGCGUUCCUAGAAGUACACGAUCGCUUCGAGAACCUGGAUCCAAAAAAAAAAAAAAACCCAC GCAAGGAUCUUCAUGUGC(SEQ ID NO: 375) GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUC (SEQ ID NO: 376)GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAAGUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUAGACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAUUACCCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCACUCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUACUUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUAG (SEQ ID NO: 377)GGGAGAAAGCUCAAGCUUGGAGCAAUGCCCGCACAUUGAGGAAACCGAGUUGCAUAUCUCAGAGUAUUGGCCCCCGUGUAGGUUAUUCUUGACAGACAGUGGAGCUUAUUCACUCCCAGGAUCCGAGUCGCAUACUACGGUACUGGUGACAGACCUAGGUCGUCAGUUGACCAGUCCGCCACUAGACGUGAGUCCGUCAAAGCAGUUAGAUGUUACACUCUAUUAGAUCUCGGAUUACAGCUGGAAGGAGCAGGAGUAGUGUUCUUGCUCUAAGUACCGAGUGUGCCCAAUACCCGAUCAGCUUAUUAACGAACGGCUCCUCCUCUUAGACUGCAGCGUAAGUGCGGAAUCUGGGGAUCAAAUUACUGACUGCCUGGAUUACCCUCGGACAUAUAACCUUGUAGCACGCUGUUGCUGUAUAGGUGACCAACGCCCACUCGAGUAGACCAGCUCUCUUAGUCCGGACAAUGAUAGGAGGCGCGGUCAAUCUACUUCUGGCUAGUUAAGAAUAGGCUGCACCGACCUCUAUAAGUAGCGUGUCCUCUAGAGCUACGCAGGUUCGCAAUAAAAGCGUUGAUUAGUGUGCAUAGAACAGACCUCUUAUUCGGUGAAACGCCAGAAUGCUAAAUUCCAAUAACUCUUCCCAAAACGCGUACGGCCGAAGACGCGCGCUUAUCUUGUGUACGUUCUCGCACAUGGAAGAAUCAGCGGGCAUGGUGGUAGGGCAAUAGGGGAGCUGGGUAGCAGCGAAAAAGGGCCCCUGCGCACGUAGCUUCGCUGUUCGUCUGAAACAACCCGGCAUCCGUUGUAGCGAUCCCGUUAUCAGUGUUAUUCUUGUGCGCACUAAGAUUCAUGGUGUAGUCGACAAUAACAGCGUCUUGGCAGAUUCUGGUCACGUGCCCUAUGCCCGGGCUUGUGCCUCUCAGGUGCACAGCGAUACUUAAAGCCUUCAAGGUACUCGACGUGGGUACCGAUUCGUGACACUUCCUAAGAUUAUUCCACUGUGUUAGCCCCGCACCGCCGACCUAAACUGGUCCAAUGUAUACGCAUUCGCUGAGCGGAUCGAUAAUA AAAGCUUGAAUU(SEQ ID NO: 378) GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUC (R 722 SEQ ID NO: 379)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUA (SEQ ID NO: 380)GGGAGAAAGCUCAAGCUUAUCCAAGUAGGCUGGUCACCUGUACAACGUAGCCGGUAUUUUUUUUUUUUUUUUUUUUUUGACCGUCUCAAGGUCCAAGUUAGUCUGCCUAUAAAGGUGCGGAUCCACAGCUGAUGAAAGACUUGUGCGGUACGGUUUAAUCUCCCCUUUUUUUUUUUUUUUUUUUUAGUAAAUGCGUCUACUGAAUCCAGCGAUGAUGCUGGCCCAGAUCUUCGACCACAAGUGCAUAUAGUAGUCAUCGAGGGUCGCCUUUUUUUUUUUUUUUUUUUUUUUGGCCCAGUUCUGAGACUUCGCUAGAGACUACAGUUACAGCUGCAGUAGUAACCACUGCGGCUAUUGCAGGAAAUCCCGUUCAGGUUUUUUUUUUUUUUUUUUUUUCCGCUCACUAUGAUUAAGAACCAGGUGGAGUGUCACUGCUCUCGAGGUCUCACGAGAGCGCUCGAUACAGUCCUUGGAAGAAUCUUUUUUUUUUUUUUUUUUUUUUGUGCGACGAUCACAGAGAACUUCUAUUCAUGCAGGUCUGCUCUAGAACGAACUGACCUGACGCCUGAACUUAUGAGCGUGCGUAUUUUUUUUUUUUUUUUUUUUUUUCCUCCCAACAAAUGUCGAUCAAUAGCUGGGCUGUUGGAGACGCGUCAGCAAAUGCCGUGGCUCCAUAGGACGUGUAGACUUCUAUUUUUUUUUUUUUUUUUUUUUCCCGGGACCACAAAUAAUAUUCUUGCUUGGUUGGGCGCAAGGGCCCCGUAUCAGGUCAUAAACGGGUACAUGUUGCACAGGCUCCUUUUUUUUUUUUUUUUUUUUUUUCGCUGAGUUAUUCCGGUCUCAAAAGACGGCAGACGUCAGUCGACAACACGGUCUAAAGCAGUGCUACAAUCUGCCGUGUUCGUGUUUUUUUUUUUUUUUUUUUUGUGAACCUACACGGCGUGCACUGUAGUUCGCAAUUCAUAGGGUACCGGCUCAGAGUUAUGCCUUGGUUGAAAACUGCCCAGCAUACUUUUUUUUUUUUUUUUUUUUCAUAUUCCCAUGCUAAGCAAGGGAUGCCGCGAGUCAUGU UAAGCUUGAAUU

According to another very particularly preferred embodiment, the nucleicacid molecule according to formula (V) may be selected from e.g. any ofthe following sequences:

(SEQ ID NO: 381) UAGCGAAGCUCUUGGACCUACCUUUUUUUUUUUUUUCCCUGCGUUCCUAGAAGUACACG or (SEQ ID NO: 382)UAGCGAAGCUCUUGGACCUACCUUUUUUUUUUUUUUUCCCUGCGUUCCUAGAAGUACACGAUCGCUUCGAGAACCUGGAUGGAAAAAAAAAAAAAAAGGGAC GCAAGGAUCUUCAUGUGC

In a further preferred embodiment the nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex may also occurin the form of a modified nucleic acid.

According to a first aspect, the nucleic acid molecule of the hereindefined inventive polymeric carrier cargo complex may be provided as a“stabilized nucleic acid”, preferably as a stabilized RNA or DNA, morepreferably as a RNA that is essentially resistant to in vivo degradation(e.g. by an exo- or endo-nuclease).

In this context, the nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex may contain backbonemodifications, sugar modifications or base modifications. A backbonemodification in connection with the present invention is a modificationin which phosphates of the backbone of the nucleotides contained in thenucleic acid molecule of the inventive polymeric carrier cargo complexare chemically modified. A sugar modification in connection with thepresent invention is a chemical modification of the sugar of thenucleotides of the nucleic acid molecule of the inventive polymericcarrier cargo complex. Furthermore, a base modification in connectionwith the present invention is a chemical modification of the base moietyof the nucleotides of the nucleic acid molecule of the inventivepolymeric carrier cargo complex.

According to a further aspect, the nucleic acid molecule of the hereindefined inventive polymeric carrier cargo complex can contain a lipidmodification. Such a lipid-modified nucleic acid typically comprises anucleic acid as defined herein. Such a lipid-modified nucleic acidmolecule of the inventive polymeric carrier cargo complex typicallyfurther comprises at least one linker covalently linked with thatnucleic acid molecule, and at least one lipid covalently linked with therespective linker. Alternatively, the lipid-modified nucleic acidmolecule comprises at least one nucleic acid molecule as defined hereinand at least one (bifunctional) lipid covalently linked (without alinker) with that nucleic acid molecule. According to a thirdalternative, the lipid-modified nucleic acid molecule comprises anucleic acid molecule as defined herein, at least one linker covalentlylinked with that nucleic acid molecule, and at least one lipidcovalently linked with the respective linker, and also at least one(bifunctional) lipid covalently linked (without a linker) with thatnucleic acid molecule.

The nucleic acid molecule of the inventive polymeric carrier cargocomplex may likewise be stabilized in order to prevent degradation ofthe nucleic acid molecule by various approaches, particularly, when RNAor mRNA is used as a nucleic acid molecule for the inventive purpose. Itis known in the art that instability and (fast) degradation of RNA ingeneral may represent a serious problem in the application of RNA basedcompositions. This instability of RNA is typically due to RNA-degradingenzymes, “RNAases” (ribonucleases), wherein contamination with suchribonucleases may sometimes completely degrade RNA in solution.Accordingly, the natural degradation of RNA in the cytoplasm of cells isvery finely regulated and RNase contaminations may be generally removedby special treatment prior to use of said compositions, in particularwith diethyl pyrocarbonate (DEPC). A number of mechanisms of naturaldegradation are known in this connection in the prior art, which may beutilized as well. E.g., the terminal structure is typically of criticalimportance particularly for an mRNA. As an example, at the 5′ end ofnaturally occurring mRNAs there is usually a so-called “cap structure”(a modified guanosine nucleotide), and at the 3′ end is typically asequence of up to 200 adenosine nucleotides (the so-called poly-A tail).

According to another aspect, the nucleic acid molecule of the hereindefined inventive polymeric carrier cargo complex may be modified, andthus stabilized, especially if the nucleic acid molecule is in the formof a coding nucleic acid e.g. an mRNA, by modifying the G/C content ofthe nucleic acid molecule, particularly an mRNA, preferably of thecoding region thereof.

In a particularly preferred aspect of the present invention, the G/Ccontent of the coding region of the nucleic acid molecule of the hereindefined inventive polymeric carrier cargo complex, especially if thenucleic acid molecule is in the form of an mRNA, is modified,particularly increased, compared to the G/C content of the coding regionof its particular wild type coding sequence, i.e. the unmodified mRNA.The encoded amino acid sequence of the nucleic acid sequence ispreferably not modified compared to the coded amino acid sequence of theparticular wild type mRNA.

The modification of the G/C-content of the nucleic acid molecule of theherein defined inventive polymeric carrier cargo complex, especially ifthe nucleic acid molecule is in the form of an mRNA or codes for anmRNA, is based on the fact that the sequence of any mRNA region to betranslated is important for efficient translation of that mRNA. Thus,the composition and the sequence of various nucleotides are important.In particular, sequences having an increased G (guanosine)/C (cytosine)content are more stable than sequences having an increased A(adenosine)/U (uracil) content. According to the invention, the codonsof the coding sequence or mRNA are therefore varied compared to its wildtype coding sequence or mRNA, while retaining the translated amino acidsequence, such that they include an increased amount of G/C nucleotides.In respect to the fact that several codons code for one and the sameamino acid (so-called degeneration of the genetic code), the mostfavourable codons for the stability can be determined (so-calledalternative codon usage).

Preferably, the G/C content of the coding region of the nucleic acidmolecule of the herein defined inventive polymeric carrier cargocomplex, especially if the nucleic acid is in the form of an mRNA orcodes for an mRNA, is increased by at least 7%, more preferably by atleast 15%, particularly preferably by at least 20%, compared to the G/Ccontent of the coded region of the wild type mRNA. According to aspecific aspect at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, morepreferably at least 70%, even more preferably at least 80% and mostpreferably at least 90%, 95% or even 100% of the substitutable codons inthe region coding for a protein or peptide as defined herein or itsfragment or variant thereof or the whole sequence of the wild type mRNAsequence or coding sequence are substituted, thereby increasing the G/Ccontent of said sequence.

In this context, it is particularly preferable to increase the G/Ccontent of the nucleic acid molecule of the herein defined inventivepolymeric carrier cargo complex, especially if the nucleic acid is inthe form of an mRNA or codes for an mRNA, to the maximum (i.e. 100% ofthe substitutable codons), in particular in the region coding for aprotein, compared to the wild type sequence.

According to the invention, a further preferred modification of thenucleic acid molecule of the herein defined inventive polymeric carriercargo complex, especially if the nucleic acid is in the form of an mRNAor codes for an mRNA, is based on the finding that the translationefficiency is also determined by a different frequency in the occurrenceof tRNAs in cells. Thus, if so-called “rare codons” are present in thenucleic acid molecule of the inventive polymeric carrier cargo complex,especially if the nucleic acid is in the form of an mRNA or codes for anmRNA, to an increased extent, the corresponding modified nucleic acidmolecule is translated to a significantly poorer degree than in the casewhere codons coding for relatively “frequent” tRNAs are present.

Especially if the modified nucleic acid molecule of the herein definedinventive polymeric carrier cargo complex is in the form of an mRNA orcodes for an mRNA, the coding region of the modified nucleic acid ispreferably modified compared to the corresponding region of the wildtype mRNA or coding sequence such that at least one codon of the wildtype sequence which codes for a tRNA which is relatively rare in thecell is exchanged for a codon which codes for a tRNA which is relativelyfrequent in the cell and carries the same amino acid as the relativelyrare tRNA. By this modification, the sequences of the nucleic acidmolecule of the inventive polymeric carrier cargo complex, especially ifthe nucleic acid is in the form of an mRNA or codes for an mRNA, ismodified such that codons for which frequently occurring tRNAs areavailable are inserted. In other words, according to the invention, bythis modification all codons of the wild type sequence which code for atRNA which is relatively rare in the cell can in each case be exchangedfor a codon which codes for a tRNA which is relatively frequent in thecell and which, in each case, carries the same amino acid as therelatively rare tRNA.

Which tRNAs occur relatively frequently in the cell and which, incontrast, occur relatively rarely is known to a person skilled in theart; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. Thecodons which use for the particular amino acid the tRNA which occurs themost frequently, e.g. the Gly codon, which uses the tRNA which occursthe most frequently in the (human) cell, are particularly preferred.

According to the invention, it is particularly preferable to link thesequential G/C content which is increased, in particular maximized, inthe modified nucleic acid molecule of the herein defined inventivepolymeric carrier cargo complex, especially if the nucleic acid is inthe form of an mRNA or codes for an mRNA, with the “frequent” codonswithout modifying the amino acid sequence of the protein encoded by thecoding region of the nucleic acid molecule. This preferred aspect allowsprovision of a particularly efficiently translated and stabilized(modified) nucleic acid molecule of the inventive polymeric carriercargo complex, especially if the nucleic acid is in the form of an mRNAor codes for an mRNA.

According to a further preferred aspect of the invention, the nucleicacid molecule of the inventive polymeric carrier cargo complex asdefined herein, especially if the nucleic acid is in the form of acoding nucleic acid molecule, preferably has at least one 5′ and/or 3′stabilizing sequence. These stabilizing sequences in the 5′ and/or 3′untranslated regions have the effect of increasing the half-life of thenucleic acid in the cytosol. These stabilizing sequences can have 100%sequence identity to naturally occurring sequences which occur inviruses, bacteria and eukaryotes, but can also be partly or completelysynthetic. The untranslated sequences (UTR) of the (alpha-)globin gene,e.g. from Homo sapiens or Xenopus laevis may be mentioned as an exampleof stabilizing sequences which can be used in the present invention fora stabilized nucleic acid. Another example of a stabilizing sequence hasthe general formula (C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC (SEQ ID NO:383), which is contained in the 3′UTR of the very stable RNA which codesfor (alpha-)globin, type(I)-collagen, 15-lipoxygenase or for tyrosinehydroxylase (cf. Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94:2410 to 2414). Such stabilizing sequences can of course be usedindividually or in combination with one another and also in combinationwith other stabilizing sequences known to a person skilled in the art.

Nevertheless, substitutions, additions or eliminations of bases arepreferably carried out with the nucleic acid molecule of the inventivepolymeric carrier cargo complex as defined herein, especially if thenucleic acid is in the form of an mRNA, using a DNA matrix forpreparation of the nucleic acid molecule by techniques of the well knownsite directed mutagenesis or with an oligonucleotide ligation strategy(see e.g. Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, 3rd ed., Cold Spring Harbor, N.Y.,2001). In such a process, for preparation of the nucleic acid moleculeof the inventive polymeric carrier cargo complex as defined herein,especially if the nucleic acid is in the form of an mRNA, acorresponding DNA molecule may be transcribed in vitro. This DNA matrixpreferably comprises a suitable promoter, e.g. a T7 or SP6 promoter, forin vitro transcription, which is followed by the desired nucleotidesequence for the nucleic acid molecule, e.g. mRNA, to be prepared and atermination signal for in vitro transcription. The DNA molecule, whichforms the matrix of the at least one RNA of interest, may be prepared byfermentative proliferation and subsequent isolation as part of a plasmidwhich can be replicated in bacteria. Plasmids which may be mentioned assuitable for the present invention are e.g. the plasmids pT7 Ts (GenBankaccession number U26404; Lai et al., Development 1995, 121: 2349 to2360), pGEM® series, e.g. pGEM®-1 (GenBank accession number X65300; fromPromega) and pSP64 (GenBank accession number X65327); cf. also Mezei andStorts, Purification of PCR Products, in: Griffin and Griffin (ed.), PCRTechnology: Current Innovation, CRC Press, Boca Raton, Fla., 2001.

Nucleic acid molecules used according to the invention as defined hereinmay be prepared using any method known in the art, including syntheticmethods such as e.g. solid phase synthesis, as well as in vitro methods,such as in vitro transcription reactions.

According to another particularly preferred aspect, the nucleic acidmolecule of the inventive polymeric carrier cargo complex as definedherein, especially if the nucleic acid is in the form of a codingnucleic acid molecule may additionally or alternatively encode asecretory signal peptide. Such signal peptides are sequences, whichtypically exhibit a length of about 15 to 30 amino acids and arepreferably located at the N-terminus of the encoded peptide, withoutbeing limited thereto. Signal peptides as defined herein preferablyallow the transport of the protein or peptide as encoded by the nucleicacid molecule of the inventive polymeric carrier cargo complex asdefined herein, especially if the nucleic acid is in the form of anmRNA, into a defined cellular compartment, preferably the cell surface,the endoplasmic reticulum (ER) or the endosomal-lysosomal compartment.Examples of secretory signal peptide sequences as defined hereininclude, without being limited thereto, signal sequences of classical ornon-classical MHC-molecules (e.g. signal sequences of MHC I and IImolecules, e.g. of the MHC class I molecule HLA-A*0201), signalsequences of cytokines or immunoglobulins as defined herein, signalsequences of the invariant chain of immunoglobulins or antibodies asdefined herein, signal sequences of Lamp1, Tapasin, Erp57, Calreticulin,Calnexin, and further membrane associated proteins or of proteinsassociated with the endoplasmic reticulum (ER) or theendosomal-lysosomal compartment. Particularly preferably, signalsequences of MHC class I molecule HLA-A*0201 may be used according tothe present invention.

Any of the above modifications may be applied to the nucleic acidmolecule of the inventive polymeric carrier cargo complex as definedherein and further to any nucleic acid as used in the context of thepresent invention and may be, if suitable or necessary, be combined witheach other in any combination, provided, these combinations ofmodifications do not interfere with each other in the respective nucleicacid. A person skilled in the art will be able to take his choiceaccordingly.

The nucleic acid molecule of the inventive polymeric carrier cargocomplex as defined herein as well as proteins or peptides as encoded bythis nucleic acid molecule may comprise fragments or variants of thosesequences. Such fragments or variants may typically comprise a sequencehaving a sequence identity with one of the above mentioned nucleicacids, or with one of the proteins or peptides or sequences, if encodedby the at least one nucleic acid molecule, of at least 5%, 10%, 20%,30%, 40%, 50%, 60%, preferably at least 70%, more preferably at least80%, equally more preferably at least 85%, even more preferably at least90% and most preferably at least 95% or even 97%, 98% or 99%, to theentire wild type sequence, either on nucleic acid level or on amino acidlevel.

“Fragments” of proteins or peptides in the context of the presentinvention (encoded by a nucleic acid as defined herein) may comprise asequence of a protein or peptide as defined herein, which is, withregard to its amino acid sequence (or its encoded nucleic acidmolecule), N-terminally, C-terminally and/or intrasequentially truncatedcompared to the amino acid sequence of the original (native) protein (orits encoded nucleic acid molecule). Such truncation may thus occureither on the amino acid level or correspondingly on the nucleic acidlevel. A sequence identity with respect to such a fragment as definedherein may therefore preferably refer to the entire protein or peptideas defined herein or to the entire (coding) nucleic acid molecule ofsuch a protein or peptide. Likewise, “fragments” of nucleic acids in thecontext of the present invention may comprise a sequence of a nucleicacid as defined herein, which is, with regard to its nucleic acidmolecule 5′-, 3′- and/or intrasequentially truncated compared to thenucleic acid molecule of the original (native) nucleic acid molecule. Asequence identity with respect to such a fragment as defined herein maytherefore preferably refer to the entire nucleic acid as defined herein.

Fragments of proteins or peptides in the context of the presentinvention (e.g. as encoded by the nucleic acid molecule of the inventivepolymeric carrier cargo complex) may furthermore comprise a sequence ofa protein or peptide as defined herein, which has a length of about 6 toabout 20 or even more amino acids, e.g. fragments as processed andpresented by MHC class I molecules, preferably having a length of about8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12amino acids), or fragments as processed and presented by MHC class IImolecules, preferably having a length of about 13 or more amino acids,e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, whereinthese fragments may be selected from any part of the amino acidsequence. These fragments are typically recognized by T-cells in form ofa complex consisting of the peptide fragment and an MHC molecule, i.e.the fragments are typically not recognized in their native form.

Fragments of proteins or peptides as defined herein (e.g. as encoded bythe nucleic acid molecule of the inventive polymeric carrier cargocomplex) may also comprise epitopes of those proteins or peptides.Epitopes (also called “antigen determinants”) in the context of thepresent invention are typically fragments located on the outer surfaceof (native) proteins or peptides as defined herein, preferably having 5to 15 amino acids, more preferably having 5 to 12 amino acids, even morepreferably having 6 to 9 amino acids, which may be recognized byantibodies or B-cell receptors, i.e. in their native form. Such epitopesof proteins or peptides may furthermore be selected from any of theherein mentioned variants of such proteins or peptides. In this contextantigenic determinants can be conformational or discontinous epitopeswhich are composed of segments of the proteins or peptides as definedherein that are discontinuous in the amino acid sequence of the proteinsor peptides as defined herein but are brought together in thethree-dimensional structure or continuous or linear epitopes which arecomposed of a single polypeptide chain.

“Variants” of proteins or peptides as defined in the context of thepresent invention (e.g. as encoded by a nucleic acid as defined herein)may be encoded by the nucleic acid molecule of the inventive polymericcarrier cargo complex. Thereby, a protein or peptide may be generated,having an amino acid sequence which differs from the original sequencein one or more mutation(s), such as one or more substituted, insertedand/or deleted amino acid(s). Preferably, these fragments and/orvariants have the same biological function or specific activity comparedto the full-length native protein, e.g. its specific antigenic property.

“Variants” of proteins or peptides as defined in the context of thepresent invention (e.g. as encoded by a nucleic acid as defined herein)may comprise conservative amino acid substitution(s) compared to theirnative, i.e. non-mutated physiological, sequence. Those amino acidsequences as well as their encoding nucleotide sequences in particularfall under the term variants as defined herein. Substitutions in whichamino acids, which originate from the same class, are exchanged for oneanother are called conservative substitutions. In particular, these areamino acids having aliphatic side chains, positively or negativelycharged side chains, aromatic groups in the side chains or amino acids,the side chains of which can enter into hydrogen bridges, e.g. sidechains which have a hydroxyl function. This means that e.g. an aminoacid having a polar side chain is replaced by another amino acid havinga likewise polar side chain, or, for example, an amino acidcharacterized by a hydrophobic side chain is substituted by anotheramino acid having a likewise hydrophobic side chain (e.g. serine(threonine) by threonine (serine) or leucine (isoleucine) by isoleucine(leucine)). Insertions and substitutions are possible, in particular, atthose sequence positions which cause no modification to thethree-dimensional structure or do not affect the binding region.Modifications to a three-dimensional structure by insertion(s) ordeletion(s) can easily be determined e.g. using CD spectra (circulardichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORDof Polypeptides, in: Modern Physical Methods in Biochemistry, Neubergeret al. (ed.), Elsevier, Amsterdam).

Furthermore, variants of proteins or peptides as defined herein, whichmay be encoded by the nucleic acid molecule of the inventive polymericcarrier cargo complex, may also comprise those sequences, whereinnucleotides of the nucleic acid are exchanged according to thedegeneration of the genetic code, without leading to an alteration ofthe respective amino acid sequence of the protein or peptide, i.e. theamino acid sequence or at least part thereof may not differ from theoriginal sequence in one or more mutation(s) within the above meaning.

In order to determine the percentage to which two sequences areidentical, e.g. nucleic acid sequences or amino acid sequences asdefined herein, preferably the amino acid sequences encoded by a nucleicacid sequence of the polymeric carrier as defined herein or the aminoacid sequences them selves, the sequences can be aligned in order to besubsequently compared to one another. Therefore, e.g. a position of afirst sequence may be compared with the corresponding position of thesecond sequence. If a position in the first sequence is occupied by thesame component as is the case at a position in the second sequence, thetwo sequences are identical at this position. If this is not the case,the sequences differ at this position. If insertions occur in the secondsequence in comparison to the first sequence, gaps can be inserted intothe first sequence to allow a further alignment. If deletions occur inthe second sequence in comparison to the first sequence, gaps can beinserted into the second sequence to allow a further alignment. Thepercentage to which two sequences are identical is then a function ofthe number of identical positions divided by the total number ofpositions including those positions which are only occupied in onesequence. The percentage to which two sequences are identical can bedetermined using a mathematical algorithm. A preferred, but notlimiting, example of a mathematical algorithm which can be used is thealgorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul etal. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm isintegrated in the BLAST program. Sequences which are identical to thesequences of the present invention to a certain extent can be identifiedby this program.

In the inventive polymeric carrier cargo complex, the cationic componentof the polymeric carrier as defined herein and the nucleic acid cargoare typically provided in a molar ratio of about 1 to 10000, preferablyin a molar ratio of about 5 to 5000, more preferably in a molar ratio ofabout 10 to 2500, even more preferably in a molar ratio of about 25 to2000, and most preferably in a molar ratio of about 25 to 1000 ofpolymeric carrier to nucleic acid.

Furthermore, in the inventive polymeric carrier cargo complex, thecationic component of the polymeric carrier as defined herein and thenucleic acid cargo are preferably provided in an N/P-ratio of at least0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 1.5 or 2. Preferably, the N/P-ratiolies within a range of about 0.1, 0.3, 0.4, 0.5, 0.75, 1.0, 1.5 or 2 to20, preferably in a range of about 0.2 (0.5 or 0.75 or 1.0) to 12, andeven more preferably in an N/P-ratio of about 0.4 (0.75 or 1.0) to 10.In this context, the N/P ratio is a measure of the ionic charge of thecationic (side chain) component of the polymeric carrier or of thepolymeric carrier as such. In particular, if the cationic properties ofthe cationic component are generated by nitrogens (of the amino acidside chains), the N/P ratio expresses the ratio of basic nitrogen atomsto phosphate residues in the nucleotide backbone, considering that (sidechain) nitrogen atoms in the cationic component of the polymeric carriercontribute to positive charges and phosphate of the phosphate backboneof the nucleic acid contribute to the negative charge. A formula isgiven in the Examples. The N/P-ratio is defined as thenitrogen/phosphate ratio (N/P-ratio) of the entire inventive polymericcarrier cargo complex. This is typically illustrative for thecontent/amount of cationic components, in the polymeric carrier andcharacteristic for the content/amount of nucleic acids bound orcomplexed in the inventive polymeric carrier cargo complex. It may becalculated on the basis that, for example, 1 μg RNA typically containsabout 3 nmol phosphate residues, provided that RNA exhibits astatistical distribution of bases. Additionally, 1 nmol peptidetypically contains about x nmol nitrogen residues, dependent on themolecular weight and the number of its (cationic) amino acids.

In this context it is preferable that in the inventive polymeric carriercargo complex, the cationic component of the polymeric carrier asdefined herein and the nucleic acid cargo are provided in an N/P-ratioof at least about 1 or, preferably, of a range of about 1 to 20 for invitro transfection purposes.

If the expression of an encoded protein or the transcription of anencoded nucleic acid e.g. an mRNA or siRNA of the nucleic acid cargo isintended for therapeutical purposes (in vivo application) an N/P ratioof at least 0.1 (0.2, 0.3, 0.4, 0.5, 0.6), preferably of a range ofabout 0.1 (0.2, 0.3, 0.4., 0.5, or 0.6) to 1.5 is preferred. Even morepreferred is an N/P ratio range of 0.2 to 0.9 or an N/P ratio range of0.5 to 0.9. In the case that the inventive polymeric carrier cargocomplex is used for (in vivo) immunostimulation e.g. as animmunostimulating agent or adjuvant (for the purpose to induce an innateimmune response), an N/P ratio of about 0.1 to 20 is preferred, moreparticular an N/P ratio of 0.1 to 5 or 0.1 to 1.5.

In the specific case that the induction of IFN-α is intended using theinventive polymeric cargo complex as an (in vivo) immunostimulatingagent or adjuvant an N/P ratio of at least 0.1 (0.2, 0.3, 0.4, 0.5, or0.6) or an N/P ratio range of 0.1 to 1 is preferred or more preferred isan N/P ratio range of 0.2 to 0.9 or an N/P ratio range of 0.5 to 0.9.Otherwise if the induction of TNFα is intended using the inventivepolymeric cargo complex as an (in vivo) immunostimulating agent oradjuvant an N/P ratio of 1 to 20 is particularly preferred.

The N/P ratio significantly influences the surface charge of theresulting inventive polymeric carrier cargo complex. Thus it ispreferable that the resulting inventive polymeric carrier cargo complexis positively charged for in vitro transfection purposes and negativelyor neutrally charged for in vivo transfection purposes, especially ifthe expression of an encoded protein or the transcription of an encodednucleic acid of the nucleic acid cargo is intended. The surface chargeof the resulting inventive polymeric carrier cargo complex can beindicated as Zetapotential which may be measured by Dopplerelectrophoresis method using a Zetasizer Nano (Malvern Instruments,Malvern, UK).

The present invention also provides a method of preparing the inventivepolymeric carrier cargo complex as defined herein comprising followingsteps:

-   -   a) providing at least one cationic protein or peptide as defined        herein and/or at least one cationic or polycationic polymer and        optionally at least one amino acid component (AA) as defined        herein, each comprising at least one —SH moiety,    -   b) providing at least one nucleic acid molecule as defined        herein, preferably in the above mentioned ratios    -   c) mixing the components provided in steps a) and b), preferably        in a basic or neutral milieu as defined herein, preferably in        the presence of oxygen or a further starter as defined herein,        preferably at a pH, at a temperature and at time as defined        herein, and thereby condensing and thus polymerizing the        cationic components provided in step a) with each other via        disulfide bonds (in a polymerization condensation or        polycondensation) to obtain the polymeric carrier and complexing        the nucleic acid molecule provided in step b) with the cationic        components provided in step a)    -   d) optionally purifying the inventive polymeric carrier cargo        complex obtained according to step c), preferably using a method        as defined herein;    -   e) optionally lyophilization of the inventive polymeric carrier        cargo complex obtained according to step c) or d).

The method of preparing the inventive polymeric carrier cargo complex asdefined herein comprises a multi-step condensation polymerization orpolycondensation reaction via —SH moieties of the educts e.g. cationicpeptides or polymers as defined herein and optionally further amino acidcomponents (AA) in step c). The condensation polymerization orpolycondensation reaction which occurs simultaneously to thecomplexation or electrostatic binding of the nucleic acid moleculepreferably leads to the inventive polymeric carrier cargo complexwherein the polymeric carrier is a condensation polymer, wherein thesingle components are linked by disulfide bonds.

As defined herein in a step a) of the inventive method of preparing theinventive polymeric carrier cargo complex, at least one cationic orpolycationic protein or peptide as defined herein and/or at least onecationic or polycationic polymer as defined herein are provided,preferably in the ratios indicated above. These components are mixed instep c) with the nucleic acid molecule provided in step b), preferablyin a basic or neutral milieu as defined herein, preferably in thepresence of oxygen or a further starter as defined herein, preferably ata pH, and at a temperature and at a time as defined herein, and therebycondensing and thus polymerizing these components with each other viadisulfide bonds (in a polymerization condensation or polycondensation)to obtain a polymeric carrier complexed to the nucleic acid molecule asdefined herein.

According to an alternative, in step a) of the inventive method ofpreparing the inventive polymeric carrier cargo complex at least onecationic or polycationic protein or peptide and/or at least one cationicor polycationic polymer are provided as defined herein, and optionallyat least one amino acid component (AA), are provided in step a) asdefined herein, and are used for a polymerization condensation orpolycondensation and complexation reaction prior to adding the nucleicacid of step b) but using the same polymerization conditions outlinedfor step c). The polymerized polymeric carrier and the nucleic acid ofstep b) are then mixed in step c). Preferably, the components are allprovided in the ratios indicated above and mixed, preferably in a basicor neutral milieu as defined herein, preferably in the presence ofoxygen or a further starter as defined herein, preferably at a pH, at atemperature and at time as defined herein. Upon mixing and starting thereaction, the components are condensed and thus polymerized with eachother via disulfide bonds (in a polymerization condensation orpolycondensation) to obtain a polymeric carrier complexed to the nucleicacid molecule as defined herein.

In both of the above alternatives, different polymeric carriers,particularly different peptides and/or different polymers, and may beselected in the condensation polymerization as indicated above. In thiscontext, the selection of different component(s) of the polymericcarrier is typically dependent upon the desired properties of the finalpolymeric carrier and the desired cationic strength of the finalpolymeric carrier. Accordingly, the content of cationic components, mayfurthermore be “diluted” or modified in the above alternative of step a)e.g. by introducing an amino acid component (AA) as defined herein,preferably in the above defined ratios. Thereby, a modified polymericcarrier may be obtained, wherein the cationic character of theunmodified polymeric carrier typically remains in the limitations asdefined herein. The properties of the final polymeric carrier may thusbe adjusted as desired with properties of components (AA) by insertingamino acid component (AA) as defined herein in steps a).

In step c), the at least one cationic or polycationic protein or peptideas defined herein and/or at least one cationic or polycationic polymeras defined herein, and optionally at least one amino acid component (AA)and the at least one nucleic acid as defined herein, are preferablycontained in a basic or neutral milieu in the step a) of the inventivemethod of preparing the inventive polymeric carrier cargo complex. Sucha basic or neutral milieu typically exhibits a pH range of about 5 toabout 10, preferably a pH range of about 6 to about 9, more preferably apH range of about 7 to about 8, e.g. about 6.5, 7, 7.5, 8, 8.5, or 9 orany range selected from any two of these or the aforementioned values.

Furthermore, the temperature of the solution in step c) is preferably ina range of about 5° C. to about 60° C., more preferably in a range ofabout 15° C. to about 40° C., even more preferably in a range of about20° C. to about 30° C., and most preferably in a range of about 20° C.to about 25° C., e.g. about 25° C.

In step c) of the inventive method of preparing the inventive polymericcarrier cargo complex as defined herein buffers may be used as suitable.Preferred buffers may comprise, but are not limited to carbonatebuffers, borate buffers, Bicine buffer, CHES buffer, CAPS buffer,Ethanolamine containing buffers, HEPES, MOPS buffer, Phosphate buffer,PIPES buffer, Tris buffer, Tricine buffer, TAPS buffer, and/or TESbuffer as buffering agents. Particularly preferred is a carbonatebuffer.

Upon mixing the components, preferably in the presence of oxygen,preferably in the presence of a basic or neutral mileu as definedherein, the condensation polymerization or polycondensation reaction andthe complexation of the at least one nucleic acid molecule is started.For this purpose, the mixture in step c) is preferably exposed to oxygenor may be started using a further starter, e.g. a catalytic amount of anoxidizing agent, e.g. DMSO, etc. Upon start of the condensationpolymerization or polycondensation reaction of the at least one cationicor polycationic protein or peptide and/or at least one cationic orpolycationic polymer and optionally at least one amino acid component(AA) as defined herein, are condensed and thus polymerized with eachother via disulfide bonds (polymerization condensation orpolycondensation). In this reaction step a) preferably linear polymersare created using monomers with at least one reactive —SH moiety, i.e.at least one cationic or polycationic protein or peptide and/or at leastone cationic or polycationic polymer and optionally at least one aminoacid component (AA) as defined herein, each component exhibiting atleast one free —SH-moieties as defined herein, e.g. at their terminalends. However, components with more than one, preferably two free—SH-moieties may be used, which may lead to branched polymers.Simultaneously to the polymerization reaction the cationic polymers bindto the at least one nucleic acid molecule and thereby complexing it.

According to one alternative, the inventive polymeric carrier cargocomplex additionally may be modified with a component (AA) as definedherein.

According to a first example, a component (AA) (e.g. a ligand) isattached to the cationic component prior to providing the cationiccomponent in step a) via any functionality as defined herein, e.g. a —SHmoiety. This component (AA) or (e.g. a ligand) is preferably attached tothe cationic component at one terminus of these components. If theattachment is carried out via —SH bonds, the cationic components arepreferably provided with two (or even more) —SH-moieties.

The component (AA) or (e.g. a ligand) preferably carries only one —SHmoiety. In this case, one —SH moiety of the cationic component ispreferably protected in a first step using a protecting group as knownin the art. Then, the cationic component may be bound to a component Lto form a first disulfide bond via the non-protected —SH moiety. Theprotected —SH-moiety of the cationic component is then typicallydeprotected for further reactions.

Alternatively, the above mentioned component (AA) or (e.g. a ligand) maybe used in step c) to be coupled with the cationic components providedin step a) above, e.g. via disulfide bonds without blocking the free —SHmoieties. But in this context all methods known to a skilled person ordefined herein may be used to attach the component (AA) to the cationiccomponent or to the polymeric carrier.

Alternatively, a component (AA) or (e.g. a ligand) can be bound to theinventive polymeric carrier cargo complex after step c) via anyfunctionality as defined herein, e.g. a —SH moiety. In this context itis preferable that the component (AA) (e.g. a ligand) is bound via free—SH moieties of the polymeric carrier components.

According to step c) of the inventive method of preparing the inventivepolymeric carrier cargo complex as defined herein, at least one nucleicacid molecule as defined herein is mixed with the cationic componentsprovided in step b), preferably in the above mentioned ratios.Typically, in the inventive polymeric carrier cargo complex, thecationic components as defined herein, and the at least one nucleic acidmolecule are provided in a molar ratio of about 5 to 10000, preferablyin a molar ratio of about 5 to 5000, more preferably in a molar ratio ofabout 10 to 2500, even more preferably in a molar ratio of about 10 to1000 cationic polymer to nucleic acid. The N/P ratios are preferably asindicated above. In this context it is particularly preferred that theN/P ratios are selected thereby avoiding agglomeration and toxicity invivo.

In a specific embodiment, (AA) components as defined above which do notcomprise —SH moieties can be added in step c) which are therebyincorporated into the the inventive polymeric carrier cargo complexwithout polymerization by (terminal) —SH moieties. Thereby these (AA)components is/are typically not covalently linked and includednon-covalently in the inventive complex as a further component.

According to a further step d) of the inventive method of preparing theinventive polymeric carrier cargo complex as defined herein, theinventive polymeric carrier cargo complex obtained according to step c)is optionally purified. Purification may occur by using chromatographicmethods, such as HPLC, FPLC, GPS, dialysis, etc.

According to a further step e) of the inventive method of preparing theinventive polymeric carrier cargo complex as defined herein, theinventive polymeric carrier cargo complex obtained according to step c)or d) is optionally lyophilized. For this purpose any suitablecryoprotectant or lyoprotectant may be added to the inventive polymericcarrier cargo complex obtained in step c) or d).

The inventive method of preparing the inventive polymeric carrier cargocomplex as defined herein is particularly suitable to adapt the chemicalproperties of the desired inventive polymeric carrier cargo complex dueto specific selection of its components of the polymeric carrier therebyavoiding agglomeration and toxicity in vivo.

According to a further embodiment, the present invention also provides amethod for transfecting a cell, a tissue or an organism, therebyapplying or administering the inventive polymeric carrier cargo complex,particularly for therapeutic purposes. In this context, typically afterpreparing the inventive polymeric carrier cargo complex as describedabove, the inventive polymeric carrier cargo complex is preferably oradministered to a cell, a tissue or an organism, preferably in nakedform or as a pharmaceutical composition or vaccine as described herein,more preferably using any of the administration modes as describedherein. The method for transfecting a cell may be carried out in vitro,in vivo or ex vivo.

Likewise, according to another embodiment, the present invention alsorelates to the use of the inventive polymeric carrier cargo complex,particularly for therapeutic purposes, for transfecting a cell, a tissueor an organism, thereby applying or administering the inventivepolymeric carrier cargo complex as described above to a cell, a tissueor an organism, preferably in naked form or as a pharmaceuticalcomposition or vaccine as described herein, more preferably using any ofthe administration modes as described herein. The administration may becarried out in vitro, in vivo or ex vivo.

Accordingly, in a particular preferred embodiment, the present inventionalso provides a pharmaceutical composition, comprising the inventivepolymeric carrier cargo complex formed by a nucleic acid cargo asdefined herein and the polymeric carrier as defined herein. Thepharmaceutical composition optionally comprises a pharmaceuticallyacceptable carrier and/or vehicle.

As a first ingredient, the inventive pharmaceutical compositioncomprises the inventive polymeric carrier cargo complex formed by thenucleic acid cargo and the polymeric carrier as defined herein (and,optionally, (AA) component(s)).

As a second ingredient the inventive pharmaceutical composition maycomprise at least one additional pharmaceutically active component. Apharmaceutically active component in this connection is a compound thathas a therapeutic effect to heal, ameliorate or prevent a particularindication, preferably cancer diseases, autoimmune disease, allergies orinfectious diseases. Such compounds include, without implying anylimitation, peptides or proteins, preferably as defined herein, nucleicacids, preferably as defined herein, (therapeutically active) lowmolecular weight organic or inorganic compounds (molecular weight lessthan 5000, preferably less than 1000), sugars, antigens or antibodies,preferably as defined herein, therapeutic agents already known in theprior art, antigenic cells, antigenic cellular fragments, cellularfractions; cell wall components (e.g. polysaccharides), modified,attenuated or de-activated (e.g. chemically or by irradiation) pathogens(virus, bacteria etc.), adjuvants, preferably as defined herein, etc.

Furthermore, the inventive pharmaceutical composition may comprise apharmaceutically acceptable carrier and/or vehicle. In the context ofthe present invention, a pharmaceutically acceptable carrier typicallyincludes the liquid or non-liquid basis of the inventive pharmaceuticalcomposition. If the inventive pharmaceutical composition is provided inliquid form, the carrier will typically be pyrogen-free water; isotonicsaline or buffered (aqueous) solutions, e.g phosphate, citrate etc.buffered solutions. The injection buffer may be hypertonic, isotonic orhypotonic with reference to the specific reference medium, i.e. thebuffer may have a higher, identical or lower salt content with referenceto the specific reference medium, wherein preferably such concentrationsof the afore mentioned salts may be used, which do not lead to damage ofcells due to osmosis or other concentration effects. Reference media aree.g. liquids occurring in “in vivo” methods, such as blood, lymph,cytosolic liquids, or other body liquids, or e.g. liquids, which may beused as reference media in “in vitro” methods, such as common buffers orliquids. Such common buffers or liquids are known to a skilled person.Ringer-Lactate solution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well for the inventivepharmaceutical composition, which are suitable for administration to apatient to be treated. The term “compatible” as used here means thatthese constituents of the inventive pharmaceutical composition arecapable of being mixed with the inventive polymeric carrier cargocomplex as defined herein in such a manner that no interaction occurswhich would substantially reduce the pharmaceutical effectiveness of theinventive pharmaceutical composition under typical use conditions.Pharmaceutically acceptable carriers, fillers and diluents must, ofcourse, have sufficiently high purity and sufficiently low toxicity tomake them suitable for administration to a person to be treated. Someexamples of compounds which can be used as pharmaceutically acceptablecarriers, fillers or constituents thereof are sugars, such as, forexample, lactose, glucose and sucrose; starches, such as, for example,corn starch or potato starch; cellulose and its derivatives, such as,for example, sodium carboxymethylcellulose, ethylcellulose, celluloseacetate; powdered tragacanth; malt; gelatin; tallow; solid glidants,such as, for example, stearic acid, magnesium stearate; calcium sulfate;vegetable oils, such as, for example, groundnut oil, cottonseed oil,sesame oil, olive oil, corn oil and oil from theobroma; polyols, suchas, for example, polypropylene glycol, glycerol, sorbitol, mannitol andpolyethylene glycol; alginic acid.

According to a specific aspect, the inventive pharmaceutical compositionmay comprise an (additional) adjuvant. In this context, an adjuvant maybe understood as any compound, which is suitable to initiate or increasean immune response of the innate immune system, i.e. a non-specificimmune response. With other words, when administered, the inventivepharmaceutical composition typically elicits an innate immune responsedue to the adjuvant, optionally contained therein. Such an adjuvant maybe selected from any adjuvant known to a skilled person and suitable forthe present case, i.e. supporting the induction of an innate immuneresponse in a mammal.

The inventive pharmaceutical composition may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term parenteralas used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-nodal, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional, intracranial, transdermal, intradermal,intrapulmonal, intraperitoneal, intracardial, intraarterial, andsublingual injection or infusion techniques.

Preferably, the inventive pharmaceutical composition may be administeredby parenteral injection, more preferably by subcutaneous, intravenous,intramuscular, intra-articular, intra-nodal, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional, intracranial,transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial,intraarterial, and sublingual injection or via infusion techniques.Particularly preferred is intradermal and intramuscular injection.Sterile injectable forms of the inventive pharmaceutical compositionsmay be aqueous or oleaginous suspension. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents that arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation of the inventivepharmaceutical composition.

The inventive pharmaceutical composition as defined herein may also beadministered orally in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient, i.e. the inventivepolymeric carrier cargo complex, is combined with emulsifying andsuspending agents. If desired, certain sweetening, flavoring or coloringagents may also be added.

The inventive pharmaceutical composition may also be administeredtopically, especially when the target of treatment includes areas ororgans readily accessible by topical application, e.g. includingdiseases of the skin or of any other accessible epithelial tissue.Suitable topical formulations are readily prepared for each of theseareas or organs. For topical applications, the inventive pharmaceuticalcomposition may be formulated in a suitable ointment, containing theinventive polymeric carrier cargo complex suspended or dissolved in oneor more carriers. Carriers for topical administration include, but arenot limited to, mineral oil, liquid petrolatum, white petrolatum,propylene glycol, polyoxyethylene, polyoxypropylene compound,emulsifying wax and water. Alternatively, the inventive pharmaceuticalcomposition can be formulated in a suitable lotion or cream. In thecontext of the present invention, suitable carriers include, but are notlimited to, mineral oil, sorbitan monostearate, polysorbate 60, cetylesters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol andwater.

The inventive pharmaceutical composition typically comprises a “safe andeffective amount” of the components of the inventive pharmaceuticalcomposition, particularly of the inventive polymeric carrier cargocomplex as defined herein or the nucleic acid as such. As used herein, a“safe and effective amount” means an amount of the inventive polymericcarrier cargo complex as such that is sufficient to significantly inducea positive modification of a disease or disorder as defined herein. Atthe same time, however, a “safe and effective amount” is small enough toavoid serious side-effects and to permit a sensible relationship betweenadvantage and risk. The determination of these limits typically lieswithin the scope of sensible medical judgment. A “safe and effectiveamount” of the components of the inventive pharmaceutical composition,particularly of the inventive polymeric carrier cargo complex as definedherein, will furthermore vary in connection with the particularcondition to be treated and also with the age and physical condition ofthe patient to be treated, the body weight, general health, sex, diet,time of administration, rate of excretion, drug combination, theactivity of the inventive polymeric carrier cargo complex, the severityof the condition, the duration of the treatment, the nature of theaccompanying therapy, of the particular pharmaceutically acceptablecarrier used, and similar factors, within the knowledge and experienceof the accompanying doctor. The inventive pharmaceutical composition maybe used for human and also for veterinary medical purposes, preferablyfor human medical purposes, as a pharmaceutical composition in generalor as a vaccine, immunostimulating agent or adjuvant.

According to a particular preferred embodiment, the inventivepharmaceutical composition (or the inventive polymeric carrier cargocomplex) may be provided or used as an immunostimulating agent. In thiscontext, the inventive pharmaceutical composition is preferably asdefined above. More preferably, the nucleic acid of the inventivepolymeric carrier cargo complex, preferably contained in thepharmaceutical composition, is typically an immunostimulatory nucleicacid as defined herein, e.g. a CpG-DNA or an immunostimulatory RNA(isRNA). Alternatively or additionally, the nucleic acid of theinventive polymeric carrier cargo complex, preferably contained in thepharmaceutical composition, is a coding nucleic acid as defined herein,preferably a cDNA or an mRNA, more preferably encoding an adjuvantprotein preferably as defined herein.

In a specific embodiment in this context, it is preferred that anadjuvant protein is a component of the inventive polymeric carrier cargocomplex and, preferably, of the polymeric carrier.

According to an even more preferred embodiment, the inventivepharmaceutical composition (or the inventive polymeric carrier cargocomplex) may be provided or used as an adjuvant. In this context, theadjuvant is preferably defined as the inventive pharmaceuticalcomposition above. More preferably, the nucleic acid of the inventivepolymeric carrier cargo complex, preferably contained in the adjuvant,is typically an immunostimulatory nucleic acid as defined herein, e.g. aCpG-DNA or an immunostimulatory RNA (isRNA). Alternatively oradditionally, the nucleic acid of the inventive polymeric carrier cargocomplex, preferably contained in the adjuvant, is a coding nucleic acidas defined herein, preferably a cDNA or an mRNA, more preferablyencoding an adjuvant protein, preferably as defined herein. Theinventive polymeric carrier cargo complex, preferably contained in theadjuvant, typically initiates an innate immune response in the patientto be treated. Such an adjuvant may be utilized in any accompanyingtherapy, with any known vaccine or any further (known) therapeuticagent, preferably prior to, concurrent with or subsequent toadministration of the main therapy, prior to, concurrent with orsubsequent to administration of a further (known) vaccine or a (known)further therapeutic agent.

The inventive polymeric carrier cargo complex or the inventivepharmaceutical composition as defined herein provided or used as anadjuvant is preferably capable of triggering a non-antigen-specific,(innate) immune reaction (as provided by the innate immune system),preferably in an immunostimulating manner. An immune reaction cangenerally be brought about in various ways. An important factor for asuitable immune response is the stimulation of different T-cellsub-populations. T-lymphocytes typically differentiate into twosub-populations, the T-helper 1 (Th1) cells and the T-helper 2 (Th2)cells, with which the immune system is capable of destroyingintracellular (Th1) and extracellular (Th2) pathogens (e.g. antigens).The two Th cell populations differ in the pattern of effector proteins(cytokines) produced by them. Thus, Th1 cells assist the cellular immuneresponse by activation of macrophages and cytotoxic T-cells. Th2 cells,on the other hand, promote the humoral immune response by stimulation ofB-cells for conversion into plasma cells and by formation of antibodies(e.g. against antigens). The Th1/Th2 ratio is therefore of greatimportance in the immune response. In connection with the presentinvention, the Th1/Th2 ratio of the immune response is preferablydisplaced by the immune-stimulating agent, namely the inventivepolymeric carrier cargo complex in the direction towards the cellularresponse, that is to say the Th1 response, and a predominantly cellularimmune response is thereby induced. As defined above, the inventivepolymeric carrier cargo complex exerts by itself an unspecific innateimmune response, which allows the inventive polymeric carrier cargocomplex be used as such (without adding another pharmaceutically activecomponent) as an immunostimulating agent. If administered together withanother pharmaceutically active component, preferably a specificallyimmunogenic component, preferably an antigen, the nucleic acid of theinvention serves as an adjuvant supporting the specific adaptive immuneresponse elicited by the other pharmaceutically active component e.g. anantigen.

Determination of the (Innate) Immunostimulatory or Adjuvant Capacity ofan Inventive Compound or an Inventive Complex:

For the determination of the immunostimulatory capacity of an inventivecompound or an inventive complex several methods are known in the artand may be used. E.g., in vitro methods are advantageous to screen forcompounds as to their capacity to induce cytokines, which are(exclusively or at least typically) part of the innate immune system andthereby (as an additional arm of the immune system) typically improvethe induction of an antigen-specific immune response caused by anantigen. For this purpose, e.g. PBMCs may be isolated from blood samplesand stimulated with the particular compound or complex. Afterincubation, secretion of the desired cytokines (e.g. as a reaction of anactivation of the PAMP receptors) being typically part of the innateimmune system (and not of the antigen-specific immune system) isdetermined by ELISA. These selected cytokines may be used in the art asdeterminants of the induction of an innate immune response in the body.In this context, the secretion of TNF-alpha and IFN-alpha is preferablymeasured to determine the unspecific (innate immune response) evoked bya compound or complex. Especially, IFN-alpha plays an important role inthe induction of an unspecific immune response after viral infection.Accordingly, it is particularly preferred that the the immunostimulatorycompound or complex, which shall be identified by the screening assay,induces the secretion of e.g. IFN-alpha. Such a compound or complex maythen be applied e.g. for the use as an immunostimulating agent(triggering the unspecific (innate) immune response) in vaccinationtherapies.

IFN-alpha is part of the family of type I interferons. Type Iinterferons (IFN) are pleiotropic cytokines that are essential forsupporting anti-viral immune responses. They induce apoptosis ofvirus-infected cells and cellular resistance to viral infection, inaddition to activating natural killer (NK) and T cells. Type Iinterferons have effects on a large set of cytokines and chemokines thati.a. influence immunocyte maturation, homing, effector functions andapoptosis. Typically, a major role of IFN-α/β is the induction of apriming state affecting the production and regulation of othermediators, including cytokines. For example, IFN-α/β signalingupregulates IFN-γ production by dendritic cells (DCs) and T cells andthereby favours the induction and maintenance of Th1 cells. Shifting ofan immune response in direction of a Th1 immune response may becomeimportant, once protein or peptide vaccines are used, because thesevaccines usually induce a Th2-based immune response which consequentlyprevents the induction of cytotoxic T cells.

Therefore, it is preferred that a compound or complex to be used as anadjuvant may preferably have the property of shifting anantigen-specific immune response caused by a vaccine to a Th1-basedimmune response. The direction of an immune response induced by avaccine is usually measured by determination of the induction of severalsubtypes of antigen-specific antibodies and the induction ofantigen-specific cytotoxic CD8+ T cells. In this context, the subtypeantibody IgG1 represents the induction of a Th2-based immune responseand the induction of the subtype antibody IgG2a and the induction ofcytotoxic T cells represent the induction of a Th1-based immuneresponse. The induction of antigen-specific antibodies is determined bymeasurement of the antibody titer in the blood of the vaccine by ELISA.The induction of antigen-specific cytotoxic T cells is determined bymeasurement of IFN-gamma secretion in splenocytes after stimulation withantigen-specific peptides by ELISPOT. In this context, the induction ofIFN-gamma secretion proves that antigen-specific cytotoxic T cells arepresent in the spleen which can specifically attack cells which presentepitopes of the antigen on MHC I molecules on their surface.

Thus, for the determination of beneficial properties of an adjuvant invivo vaccinations are performed. Therewith, it is possible to find out,if the adjuvant or immunostimulatory compound or complex improves anantigen-specific immune response caused by the vaccine and, furthermore,if it can shift an antigen-specific immune response in the desireddirection to display adjuvant properties. Particularly, in the inductionof an anti-tumoral immune response the induction of a Th1-shifted immuneresponse, especially the induction of cytotoxic T cells plays a majorrole, because the induction of antigen-specific cytotoxic T cellsrepresents an indispensable prerequisite for the successful combat of atumour.

Accordingly, the methods to screen for compound or complexes whichactually exhibit properties as immunostimulating agents and/or adjuvantsare well known in the art and may readily be applied e.g. by ELISA testsmeasuring the immune response elicited by the testedcompounds/complexes.

According to another particularly preferred embodiment, the inventivepharmaceutical composition (or the inventive polymeric carrier cargocomplex) may be provided or used as a vaccine.

In this context, the vaccine is preferably defined as an adjuvant or asan inventive pharmaceutical composition as disclosed above. Morepreferably, the nucleic acid of the inventive polymeric carrier cargocomplex, preferably contained such a vaccine, may be any nucleic acid asdefined above, preferably an immunostimulatory nucleic acid as definedherein, e.g. a CpG-DNA or an immunostimulatory RNA (isRNA).Alternatively or additionally, the nucleic acid of the inventivepolymeric carrier cargo complex, preferably contained in the vaccine, isa coding nucleic acid as defined herein, preferably a cDNA or an mRNA,more preferably encoding an adjuvant protein, preferably as definedherein. Alternatively or additionally, the nucleic acid of the inventivepolymeric carrier cargo complex, preferably contained in the vaccine, isa coding nucleic acid as defined herein, preferably a cDNA or an mRNA,more preferably encoding an antigen, preferably as defined herein.Furthermore, particularly, if the nucleic acid of the inventivepolymeric carrier cargo complex does not encode an antigen, theinventive vaccine may contain an antigen, preferably as defined above,either as a protein or peptide or encoded by a nucleic acid, orantigenic cells, antigenic cellular fragments, cellular fractions; cellwall components (e.g. polysaccharides), modified, attenuated orde-activated (e.g. chemically or by irradiation) pathogens (virus,bacteria etc.).

According to a first aspect such an inventive vaccine is typicallycomposed like the inventive adjuvant and preferably supports or elicitsan innate immune response of the immune system of a patient to betreated, if an immunostimulatory nucleic acid is used as the nucleicacid molecule of the inventive polymeric carrier cargo complex.

According to a second aspect the inventive vaccine may elicit anadaptive immune response, preferably, if the nucleic acid of theinventive polymeric carrier cargo complex as defined herein encodes anantigen as defined herein, suitable to elicit an adaptive immuneresponse. Alternatively, this antigen can be in form of a peptide, aprotein or an epitope or may be provided as an additional nucleic acidencoding said antigen. The antigen may also be a component of theinventive polymeric carrier, e.g. as a (AA) component, as definedherein.

The inventive vaccine, immunostimulating agent or adjuvant may alsocomprise a pharmaceutically acceptable carrier, adjuvant, and/or vehicleas defined herein for the inventive pharmaceutical composition. In thespecific context of the inventive vaccine, the choice of apharmaceutically acceptable carrier is determined in principle by themanner in which the inventive vaccine is administered. The inventivevaccine can be administered, for example, systemically or locally.Routes for systemic administration in general include, for example,transdermal, oral, parenteral routes, including subcutaneous,intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal administration routes.Routes for local administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, and sublingual injections. More preferably,vaccines may be administered by an intradermal, subcutaneous, orintramuscular route. Inventive vaccines are therefore preferablyformulated in liquid (or sometimes in solid) form. The suitable amountof the inventive vaccine to be administered can be determined by routineexperiments with animal models. Such models include, without implyingany limitation, rabbit, sheep, mouse, rat, dog and non-human primatemodels. Preferred unit dose forms for injection include sterilesolutions of water, physiological saline or mixtures thereof. The pH ofsuch solutions should be adjusted to about 7.4. Suitable carriers forinjection include hydrogels, devices for controlled or delayed release,polylactic acid and collagen matrices. Suitable pharmaceuticallyacceptable carriers for topical application include those which aresuitable for use in lotions, creams, gels and the like. If the inventivevaccine is to be administered orally, tablets, capsules and the like arethe preferred unit dose form. The pharmaceutically acceptable carriersfor the preparation of unit dose forms which can be used for oraladministration are well known in the prior art. The choice thereof willdepend on secondary considerations such as taste, costs and storability,which are not critical for the purposes of the present invention, andcan be made without difficulty by a person skilled in the art.

The inventive vaccine, immunostimulating agent or adjuvant canadditionally contain one or more auxiliary substances in order toincrease its immunogenicity or immunostimulatory capacity, if desired. Asynergistic action of the inventive polymeric carrier cargo complex asdefined herein and of an auxiliary substance, which may be optionallycontained in the inventive vaccine, immunostimulating agent or adjuvantas defined herein, is preferably achieved thereby. Depending on thevarious types of auxiliary substances, various mechanisms can come intoconsideration in this respect. For example, compounds that permit thematuration of dendritic cells (DCs), for example lipopolysaccharides,TNF-alpha or CD40 ligand, form a first class of suitable auxiliarysubstances. In general, it is possible to use as auxiliary substance anyagent that influences the immune system in the manner of a “dangersignal” (LPS, GP96, etc.) or cytokines, such as GM-CFS, which allow animmune response to be enhanced and/or influenced in a targeted manner.Particularly preferred auxiliary substances are cytokines, such asmonokines, lymphokines, interleukins or chemokines, that further promotethe innate immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, INF-alpha, IFN-beta,INF-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors,such as hGH.

Further additives which may be included in the inventive vaccine,immunostimulating agent or adjuvant are emulsifiers, such as, forexample, Tween®; wetting agents, such as, for example, sodium laurylsulfate; colouring agents; taste-imparting agents, pharmaceuticalcarriers; tablet-forming agents; stabilizers; antioxidants;preservatives.

The inventive vaccine, immunostimulating agent or adjuvant can alsoadditionally contain any further compound, which is known to beimmunostimulating due to its binding affinity (as ligands) to humanToll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, or due to its binding affinity (as ligands) to murineToll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12 or TLR13.

The inventive vaccine, immunostimulating agent or adjuvant can alsoadditionally or alternatively contain an immunostimulatory RNA, i.e. anRNA derived from an immunostimulatory RNA, which triggers or increasesan (innate) immune response. Preferably, such an immunostimulatory RNAmay be in general be as defined hereinbefore.

Another class of compounds, which may be added to an inventive vaccine,immunostimulating agent or adjuvant in this context, may be CpG nucleicacids, in particular CpG-RNA or CpG-DNA. A CpG-RNA or CpG-DNA can be asingle-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA),a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (dsCpG-RNA). The CpG nucleic acid is preferably in the form of CpG-RNA,more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA). TheCpG nucleic acid preferably contains at least one or more (mitogenic)cytosine/guanine dinucleotide sequence(s) (CpG motif(s)). According to afirst preferred alternative, at least one CpG motif contained in thesesequences, that is to say the C (cytosine) and the G (guanine) of theCpG motif, is unmethylated. All further cytosines or guanines optionallycontained in these sequences can be either methylated or unmethylated.According to a further preferred alternative, however, the C (cytosine)and the G (guanine) of the CpG motif can also be present in methylatedform.

The present invention furthermore provides several applications and usesof the inventive polymeric carrier cargo complex as defined herein, theinventive pharmaceutical composition, the inventive immunostimulatingagent or adjuvant and the inventive vaccine comprising same or of kitscomprising same.

According to one specific embodiment, the present invention is directedto the first medical use of the inventive polymeric carrier cargocomplex as defined herein as a medicament, preferably as animmunostimulating agent, adjuvant or vaccine or in the field of genetherapy.

According to another embodiment, the present invention is directed tothe second medical use of the inventive polymeric carrier cargo complexas defined herein, for the treatment of diseases as defined herein,preferably to the use of inventive polymeric carrier cargo complex asdefined herein, of a pharmaceutical composition, vaccine,immunostimulating agent, adjuvant or vaccine comprising same or of kitscomprising same for the preparation of a medicament for the prophylaxis,treatment and/or amelioration of various diseases as defined herein,particularly prophylaxis, treatment and/or amelioration of such diseasesas defined herein. Preferably, the pharmaceutical composition, animmunostimulating agent, an adjuvant or a vaccine is used oradministered to a patient in need thereof for this purpose.

Preferably, diseases as mentioned herein are selected from cancer ortumour diseases, infectious diseases, preferably (viral, bacterial orprotozoological) infectious diseases, autoimmune diseases, allergies orallergic diseases, monogenetic diseases, i.e. (hereditary) diseases, orgenetic diseases in general, diseases which have a genetic inheritedbackground and which are typically caused by a single gene defect andare inherited according to Mendel's laws, cardiovascular diseases,neuronal diseases or any disease which can be influenced by the presentinvention.

Such diseases include cancer or tumor diseases, preferably selected frommelanomas, malignant melanomas, colon carcinomas, lymphomas, sarcomas,blastomas, renal carcinomas, gastrointestinal tumors, gliomas, prostatetumors, bladder cancer, rectal tumors, stomach cancer, oesophagealcancer, pancreatic cancer, liver cancer, mammary carcinomas (=breastcancer), uterine cancer, cervical cancer, acute myeloid leukaemia (AML),acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), chroniclymphocytic leukaemia (CLL), hepatomas, various virus-induced tumorssuch as, for example, papilloma virus-induced carcinomas (e.g. cervicalcarcinoma=cervical cancer), adenocarcinomas, herpes virus-induced tumors(e.g. Burkitt's lymphoma, EBV-induced B-cell lymphoma), heptatitisB-induced tumors (hepatocell carcinomas), HTLV-1- and HTLV-2-inducedlymphomas, acoustic neuroma, lung carcinomas (=lung cancer=bronchialcarcinoma), small-cell lung carcinomas, pharyngeal cancer, analcarcinoma, glioblastoma, rectal carcinoma, astrocytoma, brain tumors,retinoblastoma, basalioma, brain metastases, medulloblastomas, vaginalcancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome,meningiomas, Schneeberger disease, hypophysis tumor, Mycosis fungoides,carcinoids, neurinoma, spinalioma, Burkitt's lymphoma, laryngeal cancer,renal cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin'slymphomas, urethral cancer, CUP syndrome, head/neck tumors,oligodendroglioma, vulval cancer, intestinal cancer, colon carcinoma,oesophageal carcinoma (=oesophageal cancer), wart involvement, tumors ofthe small intestine, craniopharyngeomas, ovarian carcinoma, genitaltumors, ovarian cancer (=ovarian carcinoma), pancreatic carcinoma(=pancreatic cancer), endometrial carcinoma, liver metastases, penilecancer, tongue cancer, gall bladder cancer, leukaemia, plasmocytoma, lidtumor, prostate cancer (=prostate tumors), etc..

According to one further specific embodiment, diseases as defined hereincomprise infectious diseases, preferably (viral, bacterial orprotozoological) infectious diseases. Such infectious diseases,preferably to (viral, bacterial or protozoological) infectious diseases,are typically selected from influenza, malaria, SARS, yellow fever,AIDS, Lyme borreliosis, Leishmaniasis, anthrax, meningitis, viralinfectious diseases such as AIDS, Condyloma acuminata, hollow warts,Dengue fever, three-day fever, Ebola virus, cold, early summermeningoencephalitis (FSME), flu, shingles, hepatitis, herpes simplextype I, herpes simplex type II, Herpes zoster, influenza, Japaneseencephalitis, Lassa fever, Marburg virus, measles, foot-and-mouthdisease, mononucleosis, mumps, Norwalk virus infection, Pfeiffer'sglandular fever, smallpox, polio (childhood lameness), pseudo-croup,fifth disease, rabies, warts, West Nile fever, chickenpox, cytomegalicvirus (CMV), bacterial infectious diseases such as miscarriage (prostateinflammation), anthrax, appendicitis, borreliosis, botulism,Camphylobacter, Chlamydia trachomatis (inflammation of the urethra,conjunctivitis), cholera, diphtheria, donavanosis, epiglottitis, typhusfever, gas gangrene, gonorrhoea, rabbit fever, Heliobacter pylori,whooping cough, climatic bubo, osteomyelitis, Legionnaire's disease,leprosy, listeriosis, pneumonia, meningitis, bacterial meningitis,anthrax, otitis media, Mycoplasma hominis, neonatal sepsis(Chorioamnionitis), noma, paratyphus, plague, Reiter's syndrome, RockyMountain spotted fever, Salmonella paratyphus, Salmonella typhus,scarlet fever, syphilis, tetanus, tripper, tsutsugamushi disease,tuberculosis, typhus, vaginitis (colpitis), soft chancre, and infectiousdiseases caused by parasites, protozoa or fungi, such as amoebiasis,bilharziosis, Chagas disease, Echinococcus, fish tapeworm, fishpoisoning (Ciguatera), fox tapeworm, athlete's foot, canine tapeworm,candidosis, yeast fungus spots, scabies, cutaneous Leishmaniosis,lambliasis (giardiasis), lice, malaria, microscopy, onchocercosis (riverblindness), fungal diseases, bovine tapeworm, schistosomiasis, porcinetapeworm, toxoplasmosis, trichomoniasis, trypanosomiasis (sleepingsickness), visceral Leishmaniosis, nappy/diaper dermatitis or miniaturetapeworm.

According to another specific embodiment, diseases as defined hereincomprise autoimmune diseases as defined in the following. Autoimmunediseases can be broadly divided into systemic and organ-specific orlocalised autoimmune disorders, depending on the principalclinico-pathologic features of each disease. Autoimmune diseases may bedivided into the categories of systemic syndromes, including systemiclupus erythematosus (SLE), Sjögren's syndrome, Scleroderma, RheumatoidArthritis and polymyositis or local syndromes which may beendocrinologic (type I diabetes (Diabetes mellitus Type 1), Hashimoto'sthyroiditis, Addison's disease etc.), dermatologic (pemphigus vulgaris),haematologic (autoimmune haemolytic anaemia), neural (multiplesclerosis) or can involve virtually any circumscribed mass of bodytissue. The autoimmune diseases to be treated may be selected from thegroup consisting of type I autoimmune diseases or type II autoimmunediseases or type III autoimmune diseases or type IV autoimmune diseases,such as, for example, multiple sclerosis (MS), rheumatoid arthritis,diabetes, type I diabetes (Diabetes mellitus Type 1), chronicpolyarthritis, Basedow's disease, autoimmune forms of chronic hepatitis,colitis ulcerosa, type I allergy diseases, type II allergy diseases,type III allergy diseases, type IV allergy diseases, fibromyalgia, hairloss, Bechterew's disease, Crohn's disease, Myasthenia gravis,neurodermitis, Polymyalgia rheumatica, progressive systemic sclerosis(PSS), Reiter's syndrome, rheumatic arthritis, psoriasis, vasculitis,etc, or type II diabetes. While the exact mode as to why the immunesystem induces an immune reaction against autoantigens has not beenelucidated so far, there are several findings with regard to theetiology. Accordingly, the autoreaction may be due to a T-Cell bypass. Anormal immune system requires the activation of B-cells by T-cellsbefore the former can produce antibodies in large quantities. Thisrequirement of a T-cell can be by-passed in rare instances, such asinfection by organisms producing super-antigens, which are capable ofinitiating polyclonal activation of B-cells, or even of T-cells, bydirectly binding to the 13-subunit of T-cell receptors in a non-specificfashion. Another explanation deduces autoimmune diseases from aMolecular Mimicry. An exogenous antigen may share structuralsimilarities with certain host antigens; thus, any antibody producedagainst this antigen (which mimics the self-antigens) can also, intheory, bind to the host antigens and amplify the immune response. Themost striking form of molecular mimicry is observed in Group Abeta-haemolytic streptococci, which shares antigens with humanmyocardium, and is responsible for the cardiac manifestations ofrheumatic fever.

Additionally, according to one further specific embodiment, diseases asdefined herein comprise allergies or allergic diseases, i.e. diseasesrelated to allergies. Allergy is a condition that typically involves anabnormal, acquired immunological hypersensitivity to certain foreignantigens or allergens, such as the allergy antigens as defined herein.Such allergy antigens or allergens may be selected from allergy antigensas defined herein antigens derived from different sources, e.g. fromanimals, plants, fungi, bacteria, etc. Allergens in this context includee.g. grasses, pollens, molds, drugs, or numerous environmental triggers,etc. Allergies normally result in a local or systemic inflammatoryresponse to these antigens or allergens and lead to immunity in the bodyagainst these allergens. Without being bound to theory, severaldifferent disease mechanisms are supposed to be involved in thedevelopment of allergies. According to a classification scheme by P.Gell and R. Coombs the word “allergy” was restricted to type Ihypersensitivities, which are caused by the classical IgE mechanism.Type I hypersensitivity is characterised by excessive activation of mastcells and basophils by IgE, resulting in a systemic inflammatoryresponse that can result in symptoms as benign as a runny nose, tolife-threatening anaphylactic shock and death. Well known types ofallergies include, without being limited thereto, allergic asthma(leading to swelling of the nasal mucosa), allergic conjunctivitis(leading to redness and itching of the conjunctiva), allergic rhinitis(“hay fever”), anaphylaxis, angiodema, atopic dermatitis (eczema),urticaria (hives), eosinophilia, respiratory, allergies to insectstings, skin allergies (leading to and including various rashes, such aseczema, hives (urticaria) and (contact) dermatitis), food allergies,allergies to medicine, etc. Treatment of such allergic disorders ordiseases may occur preferably by desensitizing the immune reaction whichtriggers a specific immune response. Such a desensitizing may be carriedout by administering an effective amount of the allergen or allergicantigen encoded by the nucleic acid as defined herein, preferably, whenformulated as a pharmaceutical composition, to induce a slight immunereaction. The amount of the allergen or allergic antigen may then beraised step by step in subsequent administrations until the immunesystem of the patient to be treated tolerates a specific amount ofallergen or allergic antigen.

Additionally, diseases to be treated in the context of the presentinvention likewise include (hereditary) diseases, or genetic diseases ingeneral monogenetic diseases, i.e. (hereditary) diseases, or geneticdiseases in general. Such (mono-)genetic diseases, (hereditary)diseases, or genetic diseases in general are typically caused by geneticdefects, e.g. due to gene mutations resulting in loss of proteinactivity or regulatory mutations which do not allow transcription ortranslation of the protein. Frequently, these diseases lead to metabolicdisorders or other symptoms, e.g. muscle dystrophy. The presentinvention allows treating the following (hereditary) diseases or geneticdiseases: 3-beta-hydroxysteroid dehydrogenase deficiency (type II);3-ketothiolase deficiency; 6-mercaptopurine sensitivity; Aarskog-Scottsyndrome; Abetalipoproteinemia; Acatalasemia; Achondrogenesis;Achondrogenesis-hypochondrogenesis; Achondroplasia; Achromatopsia;Acromesomelic dysplasia (Hunter-Thompson type); ACTH deficiency;Acyl-CoA dehydrogenase deficiency (short-chain, medium chain, longchain); Adenomatous polyposis coli; Adenosin-deaminase deficiency;Adenylosuccinase deficiency; Adhalinopathy; Adrenal hyperplasia,congenital (due to 11-beta-hydroxylase deficiency; due to17-alpha-hydroxylase deficiency; due to 21-hydroxylase deficiency);Adrenal hypoplasia, congenital, with hypogonadotropic hypogonadism;Adrenogenital syndrom; Adrenoleukodystrophy; Adrenomyeloneuropathy;Afibrinogenemia; Agammaglobulinemia; Alagille syndrome; Albinism (brown,ocular, oculocutaneous, rufous); Alcohol intolerance, acute; Aldolase Adeficiency; Aldosteronism, glucocorticoid-remediable; Alexander disease;Alkaptonuria; Alopecia universalis; Alpha-1-antichymotrypsin deficiency;Alpha-methylacyl-CoA racemase deficiency; Alpha-thalassemia/mentalretardation syndrome; Alport syndrome; Alzheimer disease-1(APP-related); Alzheimer disease-3; Alzheimer disease-4; Amelogenesisimperfecta; Amyloid neuropathy (familial, several allelic types);Amyloidosis (Dutch type; Finnish type; hereditary renal; renal; senilesystemic); Amytrophic lateral sclerosis; Analbuminemia; Androgeninsensitivity; Anemia (Diamond-Blackfan); Anemia (hemolytic, due to PKdeficiency); Anemia (hemolytic, Rh-null, suppressor type); Anemia(neonatal hemolytic, fatal and nearfatal); Anemia (sideroblastic, withataxia); Anemia (sideroblastic/hypochromic); Anemia due to G6PDdeficiency; Aneurysm (familial arterial); Angelman syndrome; Angioedema;Aniridia; Anterior segment anomalies and cataract; Anterior segmentmesenchymal dysgenesis; Anterior segment mesenchymal dysgenesis andcataract; Antithrombin III deficiency; Anxiety-related personalitytraits; Apert syndrome; Apnea (postanesthetic); ApoA-I and apoC-IIIdeficiency (combined); Apolipoprotein A-II deficiency; ApolipoproteinB-100 (ligand-defective); Apparent mineralocorticoid excess(hypertension due to); Argininemia; Argininosuccinicaciduria;Arthropathy (progressive pseudorheumatoid, of childhood);Aspartylglucosaminuria; Ataxia (episodic); Ataxia with isolated vitaminE deficiency; Ataxia-telangiectasia; Atelosteogenesis II; ATP-dependentDNA ligase I deficiency; Atrial septal defect with atrioventricularconduction defects; Atrichia with papular lesions; Autism(succinylpurinemic); Autoimmune polyglandular disease, type I; Autonomicnervous system dysfunction; Axenfeld anomaly; Azoospermia;Bamforth-Lazarus syndrome; Bannayan-Zonana syndrome; Barthsyndrome;Bartter syndrome (type 2 or type 3); Basal cell carcinoma; Basal cellnevus syndrome; BCG infection; Beare-Stevenson cutis gyrata syndrome;Becker muscular dystrophy; Beckwith-Wiedemann syndrome; Bernard-Souliersyndrome (type B; type C); Bethlem myopathy; Bile acid malabsorption,primary; Biotinidase deficiency; Bladder cancer; Bleeding disorder dueto defective thromboxane A2 receptor; Bloom syndrome; Brachydactyly(type B1 or type C); Branchiootic syndrome; Branchiootorenal syndrome;Breast cancer (invasive intraductal; lobular; male, with Reifensteinsyndrome; sporadic); Breast cancer-1 (early onset); Breast cancer-2(early onset); Brody myopathy; Brugada syndrome; Brunner syndrome;Burkitt lymphoma; Butterfly dystrophy (retinal); C1q deficiency (type A;type B; type C); C1r/C1s deficiency; C1s deficiency, isolated; C2deficiency; C3 deficiency; C3b inactivator deficiency; C4 deficiency; C8deficiency, type II; C9 deficiency; Campomelic dysplasia with autosomalsex reversal; Camptodactyly-arthropathy-coxa varapericarditis syndrome;Canavan disease; Carbamoylphosphate synthetase I deficiency;Carbohydrate-deficient glycoprotein syndrome (type I; type Ib; type II);Carcinoid tumor of lung; Cardioencephalomyopathy (fatal infantile, dueto cytochrome c oxidase deficiency); Cardiomyopathy (dilated; X-linkeddilated; familial hypertrophic; hypertrophic); Carnitine deficiency(systemic primary); Carnitine-acylcarnitine translocase deficiency;Carpal tunnel syndrome (familial); Cataract (cerulean; congenital;crystalline aculeiform; juvenile-onset; polymorphic and lamellar;punctate; zonular pulverulent); Cataract, Coppock-like; CD59 deficiency;Central core disease; Cerebellar ataxia; Cerebral amyloid angiopathy;Cerebral arteriopathy with subcortical infarcts and leukoencephalopathy;Cerebral cavernous malformations-1; Cerebrooculofacioskeletal syndrome;Cerebrotendinous xanthomatosis; Cerebrovascular disease; Ceroidlipofuscinosis (neuronal, variant juvenile type, with granularosmiophilic deposits); Ceroid lipofuscinosis (neuronal-1, infantile);Ceroid-lipofuscinosis (neuronal-3, juvenile); Char syndrome;Charcot-Marie-Tooth disease; Charcot-Marie-Tooth neuropathy;Charlevoix-Saguenay type; Chediak-Higashi syndrome; Chloride diarrhea(Finnish type); Cholestasis (benign recurrent intrahepatic); Cholestasis(familial intrahepatic); Cholestasis (progressive familialintrahepatic); Cholesteryl ester storage disease; Chondrodysplasiapunctata (brachytelephalangic; rhizomelic; X-linked dominant; X-linkedrecessive; Grebe type); Chondrosarcoma; Choroideremia; Chronicgranulomatous disease (autosomal, due to deficiency of CYBA); Chronicgranulomatous disease (X-linked); Chronic granulomatous disease due todeficiency of NCF-1; Chronic granulomatous disease due to deficiency ofNCF-2; Chylomicronemia syndrome, familial; Citrullinemia; classicalCockayne syndrome-1; Cleft lip, cleft jaw, cleft palate; Cleftlip/palate ectodermal dysplasia syndrome; Cleidocranial dysplasia; CMOII deficiency; Coats disease; Cockayne syndrome-2, type B; Coffin-Lowrysyndrome; Colchicine resistance; Colon adenocarcinoma; Colon cancer;Colorblindness (deutan; protan; tritan); Colorectal cancer; Combinedfactor V and VIII deficiency; Combined hyperlipemia (familial); Combinedimmunodeficiency (X-linked, moderate); Complex I deficiency; Complexneurologic disorder; Cone dystrophy-3; Cone-rod dystrophy 3; Cone-roddystrophy 6; Cone-rod retinal dystrophy-2; Congenital bilateral absenceof vas deferens; Conjunctivitis, ligneous; Contractural arachnodactyly;Coproporphyria; Cornea plana congenita; Corneal clouding; Cornealdystrophy (Avellino type; gelatinous drop-like; Groenouw type I; latticetype I; Reis-Bucklers type); Cortisol resistance; Coumarin resistance;Cowden disease; CPT deficiency, hepatic (type I; type II); Cramps(familial, potassium-aggravated); Craniofacial-deafness-hand syndrome;Craniosynostosis (type 2); Cretinism; Creutzfeldt-Jakob disease;Crigler-Najjar syndrome; Crouzon syndrome; Currarino syndrome; Cutislaxa; Cyclic hematopoiesis; Cyclic ichthyosis; Cylindromatosis; Cysticfibrosis; Cystinosis (nephropathic); Cystinuria (type II; type III);Daltonism; Darier disease; D-bifunctional protein deficiency; Deafness,autosomal dominant 1; Deafness, autosomal dominant 11; Deafness,autosomal dominant 12; Deafness, autosomal dominant 15; Deafness,autosomal dominant 2; Deafness, autosomal dominant 3; Deafness,autosomal dominant 5; Deafness, autosomal dominant 8; Deafness,autosomal dominant 9; Deafness, autosomal recessive 1; Deafness,autosomal recessive 2; Deafness, autosomal recessive 21; Deafness,autosomal recessive 3; Deafness, autosomal recessive 4; Deafness,autosomal recessive 9; Deafness, nonsyndromic sensorineural 13;Deafness, X-linked 1; Deafness, X-linked 3; Debrisoquine sensitivity;Dejerine-Sottas disease; Dementia (familial Danish); Dementia(frontotemporal, with parkinsonism); Dent disease; Dental anomalies;Dentatorubro-pallidoluysian atrophy; Denys-Drash syndrome;Dermatofibrosarcoma protuberans; Desmoid disease; Diabetes insipidus(nephrogenic); Diabetes insipidus (neurohypophyseal); Diabetes mellitus(insulin-resistant); Diabetes mellitus (rare form); Diabetes mellitus(type II); Diastrophic dysplasia; Dihydropyrimidinuria; Dosage-sensitivesex reversal; Doyne honeycomb degeneration of retina; Dubin-Johnsonsyndrome; Duchenne muscular dystrophy; Dyserythropoietic anemia withthrombocytopenia; Dysfibrinogenemia (alpha type; beta type; gamma type);Dyskeratosis congenita-1; Dysprothrombinemia; Dystonia (DOPAresponsive);Dystonia (myoclonic); Dystonia-1 (torsion); Ectodermal dysplasia;Ectopia lentis; Ectopia pupillae; Ectrodactyly (ectodermal dysplasia,and cleft lip/palate syndrome 3); Ehlers-Danlos syndrome (progeroidform); Ehlers-Danlos syndrome (type I; type II; type III; type IV; typeVI; type VII); Elastin Supravalvar aortic stenosis; Elliptocytosis-1;Elliptocytosis-2; Elliptocytosis-3; Ellis-van Creveld syndrome;Emery-Dreifuss muscular dystrophy; Emphysema; Encephalopathy;Endocardial fibroelastosis-2; Endometrial carcinoma; Endplateacetylcholinesterase deficiency; Enhanced S-cone syndrome; Enlargedvestibular aqueduct; Epidermolysis bullosa; Epidermolysis bullosadystrophica (dominant or recessive); Epidermolysis bullosa simplex;Epidermolytic hyperkeratosis; Epidermolytic palmoplantar keratoderma;Epilepsy (generalize; juvenile; myoclonic; nocturnal frontal lobe;progressive myoclonic); Epilepsy, benign, neonatal (type1 or type2);Epiphyseal dysplasia (multiple); Episodic ataxia (type 2); Episodicataxia/myokymia syndrome; Erythremias (alpha-; dysplasia);Erythrocytosis; Erythrokeratoderma; Estrogen resistance; Exertionalmyoglobinuria due to deficiency of LDH-A; Exostoses, multiple (type 1;type 2); Exudative vitreoretinopathy, X-linked; Fabry disease; Factor Hdeficiency; Factor VII deficiency; Factor X deficiency; Factor XIdeficiency; Factor XII deficiency; Factor XIIIA deficiency; Factor XIIIBdeficiency; Familial Mediterranean fever; Fanconi anemia; Fanconi-Bickelsyndrome; Farber lipogranulomatosis; Fatty liver (acute); Favism;Fish-eye disease; Foveal hypoplasia; Fragile X syndrome; Frasiersyndrome; Friedreich ataxia; fructose-bisphosphatase Fructoseintolerance; Fucosidosis; Fumarase deficiency; Fundus albipunctatus;Fundus flavimaculatus; G6PD deficiency; GABA-transaminase deficiency;Galactokinase deficiency with cataracts; Galactose epimerase deficiency;Galactosemia; Galactosialidosis; GAMT deficiency; Gardner syndrome;Gastric cancer; Gaucher disease; Generalized epilepsy with febrileseizures plus; Germ cell tumors; Gerstmann-Straussler disease; Giantcell hepatitis (neonatal); Giant platelet disorder; Giant-cellfibroblastoma; Gitelman syndrome; Glanzmann thrombasthenia (type A; typeB); Glaucoma 1A; Glaucoma 3A; Glioblastoma multiforme;Glomerulosclerosis (focal segmental); Glucose transport defect(blood-brain barrier); Glucose/galactose malabsorption; Glucosidase Ideficiency; Glutaricaciduria (type I; type IIB; type IIC); Gluthationsynthetase deficiency; Glycerol kinase deficiency; Glycine receptor(alpha-1 polypeptide); Glycogen storage disease I; Glycogen storagedisease II; Glycogen storage disease III; Glycogen storage disease IV;Glycogen storage disease VI; Glycogen storage disease VII; Glycogenosis(hepatic, autosomal); Glycogenosis (X-linked hepatic);GM1-gangliosidosis; GM2-gangliosidosis; Goiter (adolescentmultinodular); Goiter (congenital); Goiter (nonendemic, simple); Gonadaldysgenesis (XY type); Granulomatosis, septic; Graves disease; Greigcephalopolysyndactyly syndrome; Griscelli syndrome; Growth hormonedeficient dwarfism; Growth retardation with deafness and mentalretardation; Gynecomastia (familial, due to increased aromataseactivity); Gyrate atrophy of choroid and retina with ornithinemia (B6responsive or unresponsive); Hailey-Hailey disease; Haim-Munk syndrome;Hand-foot-uterus syndrome; Harderoporphyrinuria; HDL deficiency(familial); Heart block (nonprogressive or progressive); Heinz bodyanemia; HELLP syndrome; Hematuria (familial benign); Heme oxygenase-1deficiency; Hemiplegic migraine; Hemochromotosis; Hemoglobin H disease;Hemolytic anemia due to ADA excess; Hemolytic anemia due to adenylatekinase deficiency; Hemolytic anemia due to band 3 defect; Hemolyticanemia due to glucosephosphate isomerase deficiency; Hemolytic anemiadue to glutathione synthetase deficiency; Hemolytic anemia due tohexokinase deficiency; Hemolytic anemia due to PGK deficiency;Hemolytic-uremic syndrome; Hemophagocytic lymphohistiocytosis;Hemophilia A; Hemophilia B; Hemorrhagic diathesis due to factor Vdeficiency; Hemosiderosis (systemic, due to aceruloplasminemia); Hepaticlipase deficiency; Hepatoblastoma; Hepatocellular carcinoma; Hereditaryhemorrhagic telangiectasia-1; Hereditary hemorrhagic telangiectasia-2;Hermansky-Pudlak syndrome; Heterotaxy (X-linked visceral); Heterotopia(periventricular); Hippel-Lindau syndrome; Hirschsprung disease;Histidine-rich glycoprotein Thrombophilia due to HRG deficiency; HMG-CoAlyase deficiency; Holoprosencephaly-2; Holoprosencephaly-3;Holoprosencephaly-4; Holoprosencephaly-5; Holt-Oram syndrome;Homocystinuria; Hoyeraal-Hreidarsson; HPFH (deletion type or nondeletiontype); HPRT-related gout; Huntington disease; Hydrocephalus due toaqueductal stenosis; Hydrops fetalis; Hyperbetalipoproteinemia;Hypercholesterolemia, familial; Hyperferritinemia-cataract syndrome;Hyperglycerolemia; Hyperglycinemia; Hyperimmunoglobulinemia D andperiodic fever syndrome; Hyperinsulinism; Hyperinsulinism-hyperammonemiasyndrome; Hyperkalemic periodic paralysis; Hyperlipoproteinemia;Hyperlysinemia; Hypermethioninemia (persistent, autosomal, dominant, dueto methionine, adenosyltransferase I/III deficiency);Hyperornithinemia-hyperammonemiahomocitrullinemia syndrome;Hyperoxaluria; Hyperparathyroidism; Hyperphenylalaninemia due topterin-4acarbinolamine dehydratase deficiency; Hyperproinsulinemia;Hyperprolinemia; Hypertension; Hyperthroidism (congenital);Hypertriglyceridemia; Hypoalphalipoproteinemia; Hypobetalipoproteinemia;Hypocalcemia; Hypochondroplasia; Hypochromic microcytic anemia;Hypodontia; Hypofibrinogenemia; Hypoglobulinemia and absent B cells;Hypogonadism (hypergonadotropic); Hypogonadotropic (hypogonadism);Hypokalemic periodic paralysis; Hypomagnesemia; Hypomyelination(congenital); Hypoparathyroidism; Hypophosphatasia (adult; childhood;infantile; hereditary); Hypoprothrombinemia; Hypothyroidism (congenital;hereditary congenital; nongoitrous); Ichthyosiform erythroderma;Ichthyosis; Ichthyosis bullosa of Siemens; IgG2 deficiency; Immotilecilia syndrome-1; Immunodeficiency (T-cell receptor/CD3 complex);Immunodeficiency (X-linked, with hyper-IgM); Immunodeficiency due todefect in CD3-gamma; Immunodeficiency-centromeric instabilityfacialanomalies syndrome; Incontinentia pigmenti; Insensitivity to pain(congenital, with anhidrosis); Insomnia (fatal familial); Interleukin-2receptor deficiency (alpha chain); Intervertebral disc disease;Iridogoniodysgenesis; Isolated growth hormone deficiency (IIIig typewith absent GH and Kowarski type with bioinactive GH);Isovalericacidemia; Jackson-Weiss sydnrome; Jensen syndrome; Jervell andLange-Nielsen syndrome; Joubert syndrom; Juberg-Marsidi syndrome;Kallmann syndrome; Kanzaki disease; Keratitis; Keratoderma(palmoplantar); Keratosis palmoplantaris striata I; Keratosispalmoplantaris striata II; Ketoacidosis due to SCOT deficiency; Keutelsyndrome; Klippel-Trenaurnay syndrom; Kniest dysplasia; Kostmannneutropenia; Krabbe disease; Kurzripp-Polydaktylie syndrom;Lacticacidemia due to PDX1 deficiency; Langer mesomelic dysplasia; Larondwarfism; Laurence-Moon-Biedl-Bardet syndrom; LCHAD deficiency; Lebercongenital amaurosis; Left-right axis malformation; Leigh syndrome;Leiomyomatosis (diffuse, with Alport syndrome); Leprechaunism;Leri-Weill dyschondrosteosis; Lesch-Nyhan syndrome; Leukemia (acutemyeloid; acute promyelocytic; acute T-cell lymphoblastic; chronicmyeloid; juvenile myelomonocytic; Leukemia-1 (T-cell acute lymphocytic);Leukocyte adhesion deficiency; Leydig cell adenoma; Lhermitte-Duclossyndrome; Liddle syndrome; Li-Fraumeni syndrome; Lipoamide dehydrogenasedeficiency; Lipodystrophy; Lipoid adrenal hyperplasia; Lipoproteinlipase deficiency; Lissencephaly (X-linked); Lissencephaly-1; liverGlycogen storage disease (type 0); Long QT syndrome-1; Long QTsyndrome-2; Long QT syndrome-3; Long QT syndrome-5; Long QT syndrome-6;Lowe syndrome; Lung cancer; Lung cancer (nonsmall cell); Lung cancer(small cell); Lymphedema; Lymphoma (B-cell non-Hodgkin); Lymphoma(diffuse large cell); Lymphoma (follicular); Lymphoma (MALT); Lymphoma(mantel cell); Lymphoproliferative syndrome (X-linked); Lys₁ nuricprotein intolerance; Machado-Joseph disease; Macrocytic anemiarefractory (of 5q syndrome); Macular dystrophy; Malignant mesothelioma;Malonyl-CoA decarboxylase deficiency; Mannosidosis, (alpha- or beta-);Maple syrup urine disease (type Ia; type Ib; type II); Marfan syndrome;Maroteaux-Lamy syndrome; Marshall syndrome; MASA syndrome; Mast cellleukemia; Mastocytosis with associated hematologic disorder; McArdledisease; McCune-Albright polyostotic fibrous dysplasia; McKusick-Kaufmansyndrome; McLeod phenotype; Medullary thyroid carcinoma;Medulloblastoma; Meesmann corneal dystrophy; Megaloblastic anemia-1;Melanoma; Membroproliferative glomerulonephritis; Meniere disease;Meningioma (NF2-related; SIS-related); Menkes disease; Mentalretardation (X-linked); Mephenytoin poor metabolizer; Mesothelioma;Metachromatic leukodystrophy; Metaphyseal chondrodysplasia (Murk Jansentype; Schmid type); Methemoglobinemia; Methionine adenosyltransferasedeficiency (autosomal recessive); Methylcobalamin deficiency (cbl Gtype); Methylmalonicaciduria (mutase deficiency type);Mevalonicaciduria; MHC class II deficiency; Microphthalmia (cataracts,and iris abnormalities); Miyoshi myopathy; MODY; Mohr-Tranebjaergsyndrome; Molybdenum cofactor deficiency (type A or type B);Monilethrix; Morbus Fabry; Morbus Gaucher; Mucopolysaccharidosis;Mucoviscidosis; Muencke syndrome; Muir-Torre syndrome; Mulibrey nanism;Multiple carboxylase deficiency (biotinresponsive); Multiple endocrineneoplasia; Muscle glycogenosis; Muscular dystrophy (congenitalmerosindeficient); Muscular dystrophy (Fukuyama congenital); Musculardystrophy (limb-girdle); Muscular dystrophy) Duchenne-like); Musculardystrophy with epidermolysis bullosa simplex; Myasthenic syndrome(slow-channel congenital); Mycobacterial infection (atypical, familialdisseminated); Myelodysplastic syndrome; Myelogenous leukemia; Myeloidmalignancy; Myeloperoxidase deficiency; Myoadenylate deaminasedeficiency; Myoglobinuria/hemolysis due to PGK deficiency;Myoneurogastrointestinal encephalomyopathy syndrome; Myopathy (actin;congenital; desmin-related; cardioskeletal; distal; nemaline); Myopathydue to CPT II deficiency; Myopathy due to phosphoglycerate mutasedeficiency; Myotonia congenita; Myotonia levior; Myotonic dystrophy;Myxoid liposarcoma; NAGA deficiency; Nailpatella syndrome; Nemalinemyopathy 1 (autosomal dominant); Nemaline myopathy 2 (autosomalrecessive); Neonatal hyperparathyroidism; Nephrolithiasis;Nephronophthisis (juvenile); Nephropathy (chronic hypocomplementemic);Nephrosis-1; Nephrotic syndrome; Netherton syndrome; Neuroblastoma;Neurofibromatosis (type 1 or type 2); Neurolemmomatosis; neuronal-5Ceroid-lipofuscinosis; Neuropathy; Neutropenia (alloimmune neonatal);Niemann-Pick disease (type A; type B; type C1; type D); Night blindness(congenital stationary); Nijmegen breakage syndrome; Noncompaction ofleft ventricular myocardium; Nonepidermolytic palmoplantar keratoderma;Norrie disease; Norum disease; Nucleoside phosphorylase deficiency;Obesity; Occipital hornsyndrome; Ocular albinism (Nettleship-Fallstype); Oculopharyngeal muscular dystorphy; Oguchi disease; Oligodontia;Omenn syndrome; Opitz G syndrome; Optic nerve coloboma with renaldisease; Ornithine transcarbamylase deficiency; Oroticaciduria;Orthostatic intolerance; OSMED syndrome; Ossification of posteriorlongitudinal ligament of spine; Osteoarthrosis; Osteogenesis imperfecta;Osteolysis; Osteopetrosis (recessive or idiopathic); Osteosarcoma;Ovarian carcinoma; Ovarian dysgenesis; Pachyonychia congenita(Jackson-Lawler type or Jadassohn-Lewandowsky type); Paget disease ofbone; Pallister-Hall syndrome; Pancreatic agenesis; Pancreatic cancer;Pancreatitis; Papillon-Lefevre syndrome; Paragangliomas; Paramyotoniacongenita; Parietal foramina; Parkinson disease (familial or juvenile);Paroxysmal nocturnal hemoglobinuria; Pelizaeus-Merzbacher disease;Pendred syndrome; Perineal hypospadias; Periodic fever; Peroxisomalbiogenesis disorder; Persistent hyperinsulinemic hypoglycemia ofinfancy; Persistent Mullerian duct syndrome (type II); Peters anomaly;Peutz-Jeghers syndrome; Pfeiffer syndrome; Phenylketonuria;Phosphoribosyl pyrophosphate synthetaserelated gout; Phosphorylasekinase deficiency of liver and muscle; Piebaldism; Pilomatricoma;Pinealoma with bilateral retinoblastoma; Pituitary ACTH secretingadenoma; Pituitary hormone deficiency; Pituitary tumor; Placentalsteroid sulfatase deficiency; Plasmin inhibitor deficiency; Plasminogendeficiency (types I and II); Plasminogen Tochigi disease; Plateletdisorder; Platelet glycoprotein IV deficiency; Platelet-activatingfactor acetylhydrolase deficiency; Polycystic kidney disease; Polycysticlipomembranous osteodysplasia with sclerosing leukenencephalophathy;Polydactyly, postaxial; Polyposis; Popliteal pterygium syndrome;Porphyria (acute hepatic or acute intermittent or congenitalerythropoietic); Porphyria cutanea tarda; Porphyriahepatoerythropoietic; Porphyria variegata; Prader-Willi syndrome;Precocious puberty; Premature ovarian failure; Progeria Typ I; ProgeriaTyp II; Progressive external ophthalmoplegia; Progressive intrahepaticcholestasis-2; Prolactinoma (hyperparathyroidism, carcinoid syndrome);Prolidase deficiency; Propionicacidemia; Prostate cancer; Protein Sdeficiency; Proteinuria; Protoporphyria (erythropoietic);Pseudoachondroplasia; Pseudohermaphroditism; Pseudohypoaldosteronism;Pseudohypoparathyroidism; Pseudovaginal perineoscrotal hypospadias;Pseudovitamin D deficiency rickets; Pseudoxanthoma elasticum (autosomaldominant; autosomal recessive); Pulmonary alveolar proteinosis;Pulmonary hypertension; Purpura fulminans; Pycnodysostosis;Pyropoikilocytosis; Pyruvate carboxylase deficiency; Pyruvatedehydrogenase deficiency; Rabson-Mendenhall syndrome; Refsum disease;Renal cell carcinoma; Renal tubular acidosis; Renal tubular acidosiswith deafness; Renal tubular acidosis-osteopetrosis syndrome;Reticulosis (familial histiocytic); Retinal degeneration; Retinaldystrophy; Retinitis pigmentosa; Retinitis punctata albescens;Retinoblastoma; Retinol binding protein deficiency; Retinoschisis; Rettsyndrome; Rh(mod) syndrome; Rhabdoid predisposition syndrome; Rhabdoidtumors; Rhabdomyosarcoma; Rhabdomyosarcoma (alveolar); Rhizomelicchondrodysplasia punctata; Ribbing-Syndrom; Rickets (vitaminD-resistant); Rieger anomaly; Robinow syndrome; Rothmund-Thomsonsyndrome; Rubenstein-Taybi syndrome; Saccharopinuria; Saethre-Chotzensyndrome; Salla disease; Sandhoff disease (infantile, juvenile, andadult forms); Sanfilippo syndrome (type A or type B); Schindler disease;Schizencephaly; Schizophrenia (chronic); Schwannoma (sporadic); SCID(autosomal recessive, T-negative/Bpositive type); Secretory pathwayw/TMD; SED congenita; Segawa syndrome; Selective T-cell defect; SEMD(Pakistani type); SEMD (Strudwick type); Septooptic dysplasia; Severecombined immunodeficiency (B cellnegative); Severe combinedimmunodeficiency (T-cell negative, B-cell/natural killer cell-positivetype); Severe combined immunodeficiency (Xlinked); Severe combinedimmunodeficiency due to ADA deficiency; Sex reversal (XY, with adrenalfailure); Sezary syndrome; Shah-Waardenburg syndrome; Short stature;Shprintzen-Goldberg syndrome; Sialic acid storage disorder; Sialidosis(type I or type II); Sialuria; Sickle cell anemia; Simpson-Golabi-Behmelsyndrome; Situs ambiguus; Sjogren-Larsson syndrome; Smith-Fineman-Myerssyndrome; Smith-Lemli-Opitz syndrome (type I or type II);Somatotrophinoma; Sorsby fundus dystrophy; Spastic paraplegia;Spherocytosis; Spherocytosis-1; Spherocytosis-2; Spinal and bulbarmuscular atrophy of Kennedy; Spinal muscular atrophy; Spinocerebellarataxia; Spondylocostal dysostosis; Spondyloepiphyseal dysplasia tarda;Spondylometaphyseal dysplasia (Japanese type); Stargardt disease-1;Steatocystoma multiplex; Stickler syndrome; Sturge-Weber syndrom;Subcortical laminal heteropia; Subcortical laminar heterotopia; Succinicsemialdehyde dehydrogenase deficiency; Sucrose intolerance;Sutherland-Haan syndrome; Sweat chloride elevation without CF;Symphalangism; Synostoses syndrome; Synpolydactyly; Tangier disease;Tay-Sachs disease; T-cell acute lymphoblastic leukemia; T-cellimmunodeficiency; T-cell prolymphocytic leukemia; Thalassemia (alpha- ordelta-); Thalassemia due to Hb Lepore; Thanatophoric dysplasia (types Ior II); Thiamine-responsive megaloblastic anemia syndrome;Thrombocythemia; Thrombophilia (dysplasminogenemic); Thrombophilia dueto heparin cofactor II deficiency; Thrombophilia due to protein Cdeficiency; Thrombophilia due to thrombomodulin defect; Thyroid adenoma;Thyroid hormone resistance; Thyroid iodine peroxidase deficiency; Tietzsyndrome; Tolbutamide poor metabolizer; Townes-Brocks syndrome;Transcobalamin II deficiency; Treacher Collins mandibulofacialdysostosis; Trichodontoosseous syndrome; Trichorhinophalangeal syndrome;Trichothiodystrophy; Trifunctional protein deficiency (type I or typeII); Trypsinogen deficiency; Tuberous sclerosis-1; Tuberous sclerosis-2;Turcot syndrome; Tyrosine phosphatase; Tyrosinemia; Ulnar-mammarysyndrome; Urolithiasis (2,8-dihydroxyadenine); Usher syndrome (type 1Bor type 2A); Venous malformations; Ventricular tachycardia;Virilization; Vitamin K-dependent coagulation defect; VLCAD deficiency;Vohwinkel syndrome; von Hippel-Lindau syndrome; von Willebrand disease;Waardenburg syndrome; Waardenburg syndrome/ocular albinism;Waardenburg-Shah neurologic variant; Waardenburg-Shah syndrome; Wagnersyndrome; Warfarin sensitivity; Watson syndrome; Weissenbacher-Zweymuller syndrome; Werner syndrome; Weyers acrodentaldysostosis; White sponge nevus; Williams-Beuren syndrome; Wilms tumor(type1); Wilson disease; Wiskott-Aldrich syndrome; Wolcott-Rallisonsyndrome; Wolfram syndrome; Wolman disease; Xanthinuria (type I);Xeroderma pigmentosum; X-SCID; Yemenite deaf-blind hypopigmentationsyndrome; ypocalciuric hypercalcemia (type I); Zellweger syndrome;Zlotogora-Ogur syndrome.

Diseases to be treated in the context of the present invention likewisealso include diseases which have a genetic inherited background andwhich are typically caused by a single gene defect and are inheritedaccording to Mendel's laws are preferably selected from the groupconsisting of autosomal-recessive inherited diseases, such as, forexample, adenosine deaminase deficiency, familial hypercholesterolaemia,Canavan's syndrome, Gaucher's disease, Fanconi anaemia, neuronal ceroidlipofuscinoses, mucoviscidosis (cystic fibrosis), sickle cell anaemia,phenylketonuria, alcaptonuria, albinism, hypothyreosis, galactosaemia,alpha-1-anti-trypsin deficiency, Xeroderma pigmentosum, Ribbing'ssyndrome, mucopolysaccharidoses, cleft lip, jaw, palate, Laurence MoonBiedl Bardet sydrome, short rib polydactylia syndrome, cretinism,Joubert's syndrome, type II progeria, brachydactylia, adrenogenitalsyndrome, and X-chromosome inherited diseases, such as, for example,colour blindness, e.g. red/green blindness, fragile X syndrome, musculardystrophy (Duchenne and Becker-Kiener type), haemophilia A and B, G6PDdeficiency, Fabry's disease, mucopolysaccharidosis, Norrie's syndrome,Retinitis pigmentosa, septic granulomatosis, X-SCID, ornithinetranscarbamylase deficiency, Lesch-Nyhan syndrome, or fromautosomal-dominant inherited diseases, such as, for example, hereditaryangiooedema, Marfan syndrome, neurofibromatosis, type I progeria,Osteogenesis imperfecta, Klippel-Trenaurnay syndrome, Sturge-Webersyndrome, Hippel-Lindau syndrome and tuberosis sclerosis.

The present invention also allows treatment of diseases, which have notbeen inherited, or which may not be summarized under the abovecategories. Such diseases may include e.g. the treatment of patients,which are in need of a specific protein factor, e.g. a specifictherapeutically active protein as mentioned above. This may e.g. includedialysis patients, e.g. patients which undergo a (regular) a kidney orrenal dialysis, and which may be in need of specific therapeuticallyactive proteins as defined herein, e.g. erythropoietin (EPO), etc.

Likewise, diseases in the context of the present invention may includecardiovascular diseases chosen from, without being limited thereto,coronary heart disease, arteriosclerosis, apoplexy and hypertension,etc.

Finally, diseases in the context of the present invention may be chosenfrom neuronal diseases including e.g. Alzheimer's disease, amyotrophiclateral sclerosis, dystonia, epilepsy, multiple sclerosis andParkinson's disease etc.

According to another embodiment, the present invention is directed tothe second medical use of the inventive polymeric carrier cargo complexas defined herein, for the treatment of diseases as defined herein bymeans of gene therapy.

In a further preferred embodiment, the inventive polymeric carrier cargocomplex may be used for the preparation of a pharmaceutical composition,an immunostimulating agent, an adjuvant or a vaccine.

The inventive pharmaceutical composition, immunostimulating agent,adjuvant or vaccine may furthermore be used for the treatment of adisease or a disorder as defined herein.

According to a final embodiment, the present invention also provideskits, particularly kits of parts, comprising as components alone or incombination with further ingredients at least one inventive polymericcarrier cargo complex as defined herein, at least one pharmaceuticalcomposition, immunostimulating agent, adjuvant or vaccine comprisingsame and/or kits comprising same, and optionally technical instructionswith information on the administration and dosage of the polymericcarrier molecule, the nucleic acid, the inventive polymeric carriercomplex, and/or the inventive pharmaceutical composition. Such kits,preferably kits of parts, may be applied, e.g., for any of the abovementioned applications or uses. Such kits, when occurring as a kit ofparts, may further contain each component of the inventivepharmaceutical composition, immunostimulating agent, adjuvant or vaccinein a different part of the kit.

In the present invention, if not otherwise indicated, different featuresof alternatives and embodiments may be combined with each other, wheresuitable. Furthermore, the term “comprising” shall not be construed asmeaning “consisting of”, if not specifically mentioned. However, in thecontext of the present invention, term “comprising” may be substitutedwith the term “consisting of”, where suitable.

FIGURES

The following Figures are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

FIG. 1 : shows the luciferase expression in HepG2 cells aftertransfection with polymeric carrier cargo complexes comprising 5 μg mRNAcoding for luciferase (R1180). CR₁₂C/R1180 indicates the inventivepolymeric carrier cargo complex formed by the disulfide-crosslinkedcationic peptide CR₁₂C (Cys-Arg₁₂-Cys; SEQ ID NO: 6) and the mRNA R1180as nucleic acid cargo. R₁₂/R1180 indicates a non-inventive carrier cargocomplex formed by the non-polymerizing cationic peptide R12 (Arg₁₂) andthe mRNA R1180 as nucleic acid cargo for the purpose of comparison.

-   -   The complexes contain cationic peptide and nucleic acid in a        mass ratio at 1:2 (w/w) or 2:1 (w/w) as indicated.    -   R1180 represents cells which were transfected with uncomplexed        RNA.    -   Buffer represents the negative control for non-transfected        cells.    -   After 24h the level of luciferase was quantified in the lysates        by chemoluminescence measurement of luciferin oxidation.    -   Data points indicate individual biological replicates.    -   The results firstly show that the inventive polymeric carrier        cargo complexes (CR₁₂C/R1180) induce higher levels of luciferase        expression than the non-inventive carrier cargo complexes        (R₁₂/R1180) with the non-polymerized peptide R₁₂ (SEQ ID NO:        397). And secondly it could be shown that N/P ratios higher than        1 are advantageous for in vitro transfection.

FIG. 2 : shows the (in vitro) luciferase expression in HepG2 and B 16F10cells after transfection with polymeric carrier cargo complexescomprising 5 μg mRNA coding for luciferase (R1180). CR₁₂C/R1180indicates the inventive polymeric carrier cargo complex formed by thedisulfide cross-linked cationic peptide CR₁₂C (Cys-Arg₁₂-Cys; SEQ ID NO:6) and the mRNA R1180 as nucleic acid cargo. R12/R1180 indicates anon-inventive carrier cargo complex formed by the non-polymerizingcationic peptide R₁₂(Arg₁₂) and the mRNA R1180 as nucleic acid cargo forthe purpose of comparison. CR₉C/R1180 indicates the inventive polymericcarrier cargo complex formed by the polymerized cationic peptide CR₉C(Cys-Arg₉-Cys; SEQ ID NO: 3) and the mRNA R1180 as nucleic acid cargo.

-   -   The complexes contain cationic peptide and nucleic acid in a        mass ratio at 2:1 (w/w).    -   R1180 represents cells which were transfected with uncomplexed        RNA.    -   Buffer represents the negative control for non-transfected        cells.    -   After 24h the level of luciferase was quantified in the lysates        by chemoluminescence measurement of luciferin oxidation.    -   Data points indicate individual biological replicates.    -   The results show that the inventive polymeric carrier cargo        complexes (CR₁₂C/R1180 and CR₉C/R1180) induce higher levels of        luciferase expression than the non-inventive carrier cargo        complexes (R₁₂/R1180 and R₉/1180) with the non-polymerized        peptides R9 (SEQ ID NO: 394) and R₁₂(SEQ ID NO: 397).

FIG. 3 : shows the in vivo expression of luciferase after intradermalinjection in female BALB/c mice of 5 μg mRNA coding for luciferase(R1180). CR₁₂C/R1180 indicates the inventive polymeric carrier cargocomplex formed by the disulfide-crosslinked cationic peptide CR₁₂C(Cys-Arg₁₂-Cys; SEQ ID NO: 6) and the mRNA R1180 as nucleic acid cargo.R₁₂/R1180 indicates a non-inventive carrier cargo complex formed by thenon-polymerizing cationic peptide R12 (Arg₁₂; SEQ ID NO: 397) and themRNA R1180 as nucleic acid cargo for the purpose of comparison.

The complexes contain cationic peptide and nucleic acid in a ratio of1:2 (w/w) or 2:1 as indicated.

After 24h the level of luciferase was quantified in the tissue lysatesby a chemoluminescence assay.

-   -   The results firstly show that the inventive polymeric carrier        cargo complexes (CR₁₂C/R1180) induce higher levels of luciferase        expression than the non-inventive carrier cargo complexes        (R₁₂/R1180) with the non-polymerized peptide R₁₂ (SEQ ID NO:        397; in fact no luciferase expression in the sample with the        non-polymerized peptide R₁₂could be detected). And secondly it        could be shown that N/P ratios below 1 are advantageous for in        vivo transfection.

FIG. 4 : shows the raw correlation curve of inventive polymeric carriercargo complexes formed by the disulfide-crosslinked cationic peptidesCR₁₂C (SEQ ID NO: 6) and CRTC (SEQ ID NO: 1) as carrier afterlyophilisation compared to non-inventive complexes with non-polymerizingcationic peptides as carrier (R₁₂(SEQ ID NO: 397) and R7 (SEQ ID NO:392)) by dynamic light scattering using a Zetasizer Nano (MalvernInstruments, Malvern, UK). The hydrodynamic diameters were measured withfresh prepared complexes and with reconstituted complexes afterlyophilisation The mass ratio of peptide:RNA was 1:2. As result it canbe shown that the inventive polymeric carrier cargo complexes comprisingcystein-containing peptides as cationic components which leads to apolymerization of the polymeric carrier by disulfide bonds do not changein size in contrast to the complexes formed by non-polymerizing peptideswhich increase in size and therefore are not stable during thelyophilization step. Therefore complexes with polymerized peptides aspolymeric carriers show advantageous properties for lyophilization.

FIG. 5 : shows the Zeta-potential of inventive polymeric carrier cargocomplexes formed by the disulfide-cross-linked cationic peptide CR₁₂C(SEQ ID NO: 6) and the R722 as nucleic acid cargo at different w/wratios according to the invention. As can be seen, the zeta potentialchanges from positive to negative when the w/w ratio is changed fromexcess peptide to a 1:1 ratio (peptide/RNA).

FIG. 6A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier cargo complexes formed by thedisulfide-crosslinked cationic peptide CR₁₂C (SEQ ID NO: 6) and the CpG2216 as nucleic acid cargo in a mass ratio of 1:2.5 (w/w) (CR₁₂C/CpG2216) according to the invention. As can be seen, the inventivepolymeric carrier cargo complexes lead to an increase of hIFNa cytokinerelease in hPBMCs compared to the nucleic acid cargo alone or thecationic peptide alone.

FIG. 6B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier cargo complexes formed by thedisulfide-crosslinked cationic peptide CR₁₂C (SEQ ID NO: 6) and the CpG2216 as nucleic acid cargo in a mass ratio of 1:2.5 (w/w) (CR₁₂C/CpG2216) according to the invention. As can be seen, the inventivepolymeric carrier cargo complexes do not lead to an increase in hTNFαcytokine release in hPBMCs compared to the nucleic acid cargo alone orthe cationic peptide alone.

FIG. 7A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier cargo complexes formed by thedisulfide-crosslinked cationic peptide CR₁₂C (SEQ ID NO: 6) and the mRNAR491 coding for luciferase as nucleic acid cargo in a mass ratio of 1:2(w/w) (CR₁₂C/R491) according to the invention. As can be seen, theinventive polymeric carrier cargo complexes lead to an increase of hIFNacytokine release in hPBMCs compared to the nucleic acid cargo alone orthe cationic peptide alone.

FIG. 7B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier cargo complexes formed by thedisulfide-crosslinked cationic peptide CR₁₂C (SEQ ID NO: 6) and the mRNAR491 coding for luciferase as nucleic acid cargo in a mass ratio of 1:2(w/w) (CR₁₂C/R491) according to the invention. As can be seen, theinventive polymeric carrier cargo complexes lead to an increase of hTNFαcytokine release in hPBMCs compared to the nucleic acid cargo alone orthe cationic peptide alone.

FIG. 8A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier cargo complexes formed by thedisulfide-crosslinked cationic peptide CR₁₂C (SEQ ID NO: 6) and a shortGU rich RNA oligonucleotide (short GU rich) as nucleic acid cargo in amass ratio of 1:2.5 (w/w) (CR₁₂C/short GU rich) according to theinvention. As can be seen, the inventive polymeric carrier cargocomplexes lead to an increase of hIFNa cytokine release in hPBMCscompared to the nucleic acid cargo alone or the cationic peptide alone.

FIG. 8B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier cargo complexes formed by thedisulfide-crosslinked cationic peptide CR₁₂C (SEQ ID NO: 6) and a shortGU rich RNA oligonucleotide (short GU rich) as nucleic acid cargo in amass ratio of 1:2.5 (w/w) (CR₁₂C/short GU rich) according to theinvention. As can be seen, the inventive polymeric carrier cargocomplexes lead to an increase of hTNFα cytokine release in hPBMCscompared to the nucleic acid cargo alone or the cationic peptide alone.

FIG. 9A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier cargo complexes formed by thedisulfide-crosslinked cationic peptide CR₇C (SEQ ID NO: 6) and the longnon-coding GU-rich isRNA R722 as nucleic acid cargo according to theinvention. As can be seen, the inventive polymeric carrier cargocomplexes (CR₇C/R722) lead to an increase of hIFNa cytokine release inhPBMCs compared to non-inventive carrier cargo complexes (R₇/R722)formed by the non-polymerized peptide R₇ (SEQ ID NO: 392).

FIG. 9B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier cargo complexes formed by thedisulfide-crosslinked cationic peptide CR₇C (SEQ ID NO: 6) and the longnon-coding GU-rich isRNA R722 as nucleic acid cargo according to theinvention. As can be seen, the inventive polymeric carrier cargocomplexes (CR₇C/R722) only leads to a weak increase of hTNFα cytokinerelease in hPBMCs compared to non-inventive carrier cargo complexes(R₇/R722) formed by the non-polymerized peptide R₇ (SEQ ID NO: 392).

FIG. 10A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier cargo complexes formed by thedisulfide-crosslinked cationic peptide CR₉C (SEQ ID NO: 3) and the longnon-coding GU-rich isRNA R722 as nucleic acid cargo according to theinvention. As can be seen, the inventive polymeric carrier cargocomplexes (CR₉C/R722) lead to an increase of hIFNa cytokine release inhPBMCs compared to non-inventive carrier cargo complexes (R₉/R722)formed by the non-polymerized peptide R₉ (SEQ ID NO: 394).

FIG. 10B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier cargo complexes formed by thedisulfide-crosslinked cationic peptide CR₉C (SEQ ID NO: 3) and the longnon-coding GU-rich isRNA R722 as nucleic acid cargo according to theinvention. As can be seen, the inventive polymeric carrier cargocomplexes (CR₉C/R722) do not lead to an increase of hTNFα cytokinerelease in hPBMCs compared to non-inventive carrier cargo complexes(R₉/R722) formed by the non-polymerized peptide R₉ (SEQ ID NO: 394).

FIG. 11A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier cargo complexes formed by thedisulfide-crosslinked cationic peptide CR₁₂C (SEQ ID NO: 6) and theisRNA R722 as nucleic acid cargo at different w/w ratios according tothe invention. As can be seen, the inventive polymeric carrier cargocomplexes lead to an increase in hIFNa cytokine release in hPBMCscompared to the nucleic acid cargo alone or the cationic peptide alone.

FIG. 11B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier cargo complexes formed by thedisulfide-crosslinked cationic peptide CR₁₂C (SEQ ID NO: 6) and theisRNA R722 as nucleic acid cargo at different w/w ratios according tothe invention. As can be seen, the inventive polymeric carrier cargocomplexes lead to an increase in hTNFα cytokine release in hPBMCscompared to the nucleic acid cargo alone or the cationic peptide alone.

FIG. 12A: shows the secretion of hIFNa cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier complexes formed by thecationic peptides CH₆R₄H₆C (SEQ ID NO: 388), CH₃R₄H₃C (SEQ ID NO: 390)and CHK₇HC (SEQ ID NO: 389) and the isRNA R722 as nucleic acid cargo atdifferent N/P ratios according to the invention. As can be seen, theinventive polymeric carrier cargo complexes lead to an increase in hIFNacytokine release in hPBMCs compared to the nucleic acid cargo alone orthe cationic peptide alone.

FIG. 12B: shows the secretion of hTNFα cytokine (in vitro) in hPBMCsafter stimulation with polymeric carrier complexes formed by thedisulfide-crosslinked cationic peptides CH₆R₄H₆C (SEQ ID NO: 388),CH₃R₄H₃C (SEQ ID NO: 390) and CHK₇HC (SEQ ID NO: 389) and the isRNA R722as nucleic acid cargo at different N/P ratios according to theinvention. As can be seen, the inventive polymeric carrier cargocomplexes lead to an increase in hTNFα cytokine release in hPBMCscompared to the nucleic acid cargo alone or the cationic peptide alone.Particularly inventive polymeric cargo complexes with an N/P ratiogreater or equal 1 result in TNFalpha secretion.

FIG. 13 : shows the (in vivo) effect of the addition of the inventivepolymeric carrier cargo complex formed by the disulfide-crosslinkedcationic peptide CR₁₂C (SEQ ID NO: 6) as carrier and the isRNA R722 asnucleic acid cargo to the protein vaccine Ovalbumine (OVA protein) forthe use as an adjuvant in tumour challenge experiments. As can be seen,the inventive polymeric carrier cargo complex extremely decelateratesthe tumour growth compared to the protein vaccine alone, which has noeffect on tumor growth in comparison to the buffer control.

FIG. 14 : shows the (in vivo) effect of the addition of the inventivepolymeric carrier cargo complex formed by the disulfide-crosslinkedcationic peptide CR₁₂C (SEQ ID NO: 6) as carrier and the isRNA R722 asnucleic acid cargo to the protein vaccine Ovalbumine (OVA protein) forthe use as an adjuvant on the induction of Ovalbumine-specific IgG2aantibodies. As can be seen, the inventive polymeric carrier cargocomplex strongly increases the B-cell response, which proofs thebeneficial adjuvant properties of the inventive polymeric carrier cargocomplexes, particularly in regard to the induction of a Th1-shiftedimmune response.

FIG. 15 : shows the (in vivo) effect of the addition of the inventivepolymeric carrier cargo complex formed by the disulfide-crosslinkedcationic peptide CR₁₂C (SEQ ID NO: 6) as carrier and the isRNA R722 asnucleic acid cargo to the protein vaccine Ovalbumine (OVA protein) orthe Ovalbumine-specific peptide vaccine SIINFEKL (SEQ ID NO: 535) forthe use as an adjuvant on the induction of Ovalbumine-specific cytotoxicT cells. As can be seen, the inventive polymeric carrier cargo complexstrongly increases the induction of Ovalbumin-specific cytotoxic T cellscompared to the vaccination with protein or peptide alone, which furtherproofs the beneficial adjuvant properties of the inventive polymericcarrier cargo complex, particularly in regard to the induction of aTh1-shifted immune response.

EXAMPLES

The following examples are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

1. Reagents:

Cationic Peptides as Cationic Component:

R₇: (SEQ ID NO: 392) Arg-Arg-Arg-Arg-Arg-Arg-Arg (Arg₇) CR₇C:(SEQ ID NO: 1) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (CysArg₇Cys) R₉:(SEQ ID NO: 394) Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (Arg₉) R₁₂:(SEQ ID NO: 397) Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (Arg₁₂)CR₉C: (SEQ ID NO: 3) Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys (Cys-Arg₉-Cys) CR₁₂C: (SEQ ID NO: 6)Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg (Cys-Arg₁₂-Cys)

Nucleic Acids as Cargo:

-   -   R1180: mRNA coding for luciferase (SEQ ID NO. 384)    -   R722: long non-coding is GU-rich RNA (SEQ ID NO. 385)    -   R491: mRNA coding for luciferase (SEQ ID NO. 386)    -   CpG 2216: CpG oligonucleotide (SEQ ID NO. 387)    -   Short GU rich: GU-rich RNA oligonucleotide (SEQ ID NO. 369)

2. Preparation of Nucleic Acid Sequences:

For the present examples nucleic acid sequences as indicated in example1 were prepared and used for formation of the inventive polymerizedpolymeric carrier cargo complexes or for non-polymerized carrier cargocomplexes for comparison. These polymeric carrier cargo complexes wereused for in vitro and in vivo transaction, for in vitroimmunostimulation and for particle characterizations.

According to a first preparation, the DNA sequences, coding for thecorresponding RNA sequences R1180, R722 and R491 sequences wereprepared. The sequences of the corresponding RNAs are shown in thesequence listing (SEQ ID NOs: 384, 385 and 386).

The short GU rich sequences and the CpG 2216 oligonucleotides wereprepared by automatic solid-phase synthesis by means of phosphoramiditechemistry. The sequences are shown in the sequence listing (SEQ ID NOs:387 and 369).

2. In Vitro Transcription:

The respective DNA plasmids prepared according to Example 2 for R1180,R722 and R491 were transcribed in vitro using T7-Polymerase (T7-OptimRNA Kit, CureVac, Tubingen, Germany) following the manufacturesinstructions. Subsequently the mRNA was purified using PureMessenger®(CureVac, Tubingen, Germany).

3. Synthesis of Polymeric Carrier Cargo Complexes:

The nucleic acid sequences defined above in Example 1 were mixed withthe cationic components as defined in Example 1. Therefore, theindicated amount of nucleic acid sequence was mixed with the respectivecationic component in mass ratios as indicated, thereby forming acomplex. If polymerizing cationic components were used according to thepresent invention polymerization of the cationic components took placesimultaneously to complexation of the nucleic acid cargo. Afterwards theresulting solution was adjusted with water to a final volume of 50 μland incubated for 30 min at room temperature. The different ratios ofcationic component/nucleic acid used in the experiments are shown inTable 1.

TABLE 1 Sample (cationic peptide/nucleic acid) Mass ratio N/P ratioMolar ratio CR₁₂C 1:2 0.9  44:1 (SEQ ID NO: 6)/R1180 CR₁₂C 2:1 3.6 185:1(SEQ ID NO: 6)/R1180 R₁₂ 1:2 0.7  48:1 (SEQ ID NO: 397)/R1180 R₁₂ 2:12.5 146:1 (SEQ ID NO: 397)/R1180 CR₉C 2:1 0.9  55:1 (SEQ ID NO: 3)/R1180R₉ 2:1 1.1  65:1 (SEQ ID NO: 394)/R1180 CR₇C (SEQ ID NO: 1) 1:2 0.8 70:1 R₇ (SEQ ID NO: 392) 1:2 1.0  85:1 CR₁₂C 1:2, 5 4.9  8:1 (SEQ IDNO: 6)/CpG CR₁₂C 1:2 0.9 150:1 (SEQ ID NO: 6)/R491 CR₁₂C 1:2, 5 4.9  8:1(SEQ ID NO: 6)/ short GU-rich CR₁₂C 5:1 9.6 444:1 (SEQ ID NO: 6)/R722CR₁₂C 4:1 7.6 355:1 (SEQ ID NO: 6)/R722 CR₁₂C 3:1 5.7 266:1 (SEQ ID NO:6)/R722 CR₁₂C 2:1 3.8 177:1 (SEQ ID NO: 6)/R722 CR₁₂C 1:1 1.9  88:1 (SEQID NO: 6)/R722 CR₁₂C 1:2 0.9  44:1 (SEQ ID NO: 6)/R722 CR₁₂C 1:3 0.6 29:1 (SEQ ID NO: 6)/R722 CR₁₂C 1:4 0.5  22:1 (SEQ ID NO: 6)/R722 CR₁₂C1:5 0.4  17:1 (SEQ ID NO: 6)/R722 N/P ratio = is a measure of the ioniccharge of the cationic component of the polymeric carrier or of thepolymeric carrier as such. In the case that the cationic properties ofthe cationic component are provided by nitrogen atoms the N/P ratio isthe ratio of basic nitrogen atoms to phosphate residues, consideringthat nitrogen atoms confer to positive charges and phosphate of thephosphate backbone of the nucleic acid confers to the negative charge.N/P is preferably calculated by the following formula:${N/P} = \frac{{{pmol}\lbrack{RNA}\rbrack}*{ratio}*{cationic}{AS}}{{µg}{RNA}*3*1000}$As an example the RNA R722 according to SEQ ID NO: 385 was applied,which has a molecular weight of 186 kDa. Therefore 1 μg R722 RNA confersto 5.38 pmol RNA.

4. Transfection of HepG2 and B16F10 Cells:

Polymeric carrier cargo complexes containing 5 μg mRNA coding forluciferase (R1180) were prepared as indicated in Example 3. HepG2 orB16F10 cells (150×10³/well) were seeded 1 day prior to transfection on24-well microtiter plates leading to a 70% confluence when transfectionwas carried out. For transfection 50 μl of the polymeric carrier cargocomplex solution were mixed with 250 μl serum free medium and added tothe cells (final RNA concentration: 13 μg/ml). Prior to addition of theserum free transfection solution the cells were washed gently andcarefully 2 times with 1 ml OPTI-MEM (Invitrogen) per well. Then, thetransfection solution (300 μl per well) was added to the cells and thecells were incubated for 4 h at 37° C. Subsequently 100 μl RPMI-medium(Camprex) containing 10% FCS was added per well and the cells wereincubated for additional 20 h at 37° C. The transfection solution wassucked off 24 h after transfection and the cells were lysed in 300 μllysis buffer (25 mM Tris-PO₄, 2 mM EDTA, 10% glycerol, 1% Triton-X 100,2 mM DTT). The supernatants were then mixed with luciferin buffer (25 mMGlycylglycin, 15 mM MgSO₄, 5 mM ATP, 62.5 μM luciferin) and luminiscencewas detected using a luminometer (Lumat LB 9507 (Berthold Technologies,Bad Wildbad, Germany)).

5. Expression of Luciferase In Vivo:

Polymeric carrier cargo complexes containing 5 μg mRNA coding forluciferase (R1180) were prepared as indicated in Example 3. Afterwardsthe resulting solution was adjusted with Ringer Lactate solution to afinal volume of 100 μl and incubated for 30 minutes at room temperature,yielding a solution with a 0.1 g/l concentration of polymeric carriercargo complexes. 100 μl of this solution was administrated intradermally(in the back) of 7 week old BALB/c mice. After 24h the mice weresacrificed and the samples (skin from the back) were collected, frozenat −78° C. and lysed for 3 Minutes at full speed in a tissue lyser(Qiagen, Hilden, Germany). Afterwards 600 μl of lysis buffer were addedand the resulting solutions were subjected another 6 minutes at fullspeed in the tissue lyser. After 10 minutes centrifugation at 13500 rpmat 4° C. the supernatants were mixed with luciferin buffer (25 mMGlycylglycin, 15 mM MgSO₄, 5 mM ATP, 62.5 μM luciferin) and luminiscencewas detected using a luminometer (Lumat LB 9507 (Berthold Technologies,Bad Wildbad, Germany)).

6. Cytokine Stimulation in hPBMCs:

HPBMC cells from peripheral blood of healthy donors were isolated usinga Ficoll gradient and washed subsequently with 1(PBS (phophate-bufferedsaline). The cells were then seeded on 96-well microtiter plates(200×10³/well). The hPBMC cells were incubated for 24 h with 10 μl ofthe inventive polymeric carrier cargo complex from Example 3 containingthe indicated amount of nucleic acid in X-VIVO 15 Medium (BioWhittaker).The immunostimulatory effect was measured by detecting the cytokineproduction of the hPBMCs (Tumour necrose factor alpha and Interferonalpha). Therefore, ELISA microtiter plates (Nunc MAXISORP) wereincubated over night (o/n) with binding buffer (0.02% NaN3, 15 mMNa2CO3, 15 mM NaHCO₃, pH 9.7), additionally containing a specificcytokine antibody. Cells were then blocked with 1×PBS, containing 1% BSA(bovine serum albumin). The cell supernatant was added and incubated for4 h at 37° C. Subsequently, the microtiter plate was washed with 1×PBS,containing 0.05% TWEEN-20 and then incubated with a Biotin-labelledsecondary antibody (BD Pharmingen, Heidelberg, Germany).Streptavidin-coupled horseraddish peroxidase was added to the plate.Then, the plate was again washed with 1×PBS, containing 0.05% TWEEN-20and ABTS (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) wasadded as a substrate. The amount of cytokine was determined by measuringthe absorption at 405 nm (OD 405) using a standard curve withrecombinant cytokines (BD Pharmingen, Heidelberg, Germany) with theSUNRISE ELISA-Reader from Tecan (Crailsheim, Germany).

7. Zetapotential Measurements:

The Zeta potential of the polymeric carrier cargo complexes wasevaluated by the laser Doppler electrophoresis method using a ZetasizerNano (Malvern Instruments, Malvern, UK). The measurement was performedat 25° C. and a scattering angle of 173° was used.

8. Stability of Complexes after Lyophilization

The hydrodynamic diameters of polymeric carrier cargo complexes asprepared above were measured by dynamic light scattering using aZetasizer Nano (Malvern Instruments, Malvern, UK) according to themanufacturer's instructions. The measurements were performed at 25° C.in buffer analysed by a cumulant method to obtain the hydrodynamicdiameters and polydispersity indices of the polymeric carrier cargocomplexes. Polymeric carrier cargo complexes were formed as indicated inExample 3 and the hydrodynamic diameters were measured with freshprepared complexes and with reconstituted complexes afterlyophilization.

9. Immunization Experiments:

For immunization the vaccines Ovalbumine protein (OVA) (5 μg) orOvalbumin-specific peptide SIINFEKL (SEQ ID NO: 535; 50 μg) werecombined with the inventive polymeric cargo complexes R722/CR₁₂C (SEQ IDNO: 6) (in a ratio of 2:1 w/w) (30 μg R722/15 μg CR₁₂C (SEQ ID NO: 6)).as adjuvant and injected intradermally into female C57BL/6 mice (7 miceper group for tumour challenge and 5 mice per group for detection of animmune response). The vaccination was repeated 2 times in 2 weeks. Forcomparison mice were injected without the inventive polymeric cargocomplexes.

10. Detection of an Antigen-Specific Immune Response (B-Cell ImmuneResponse):

Detection of an antigen specific immune response (B-cell immuneresponse) was carried out by detecting antigen specific antibodies.Therefore, blood samples were taken from vaccinated mice 5 days afterthe last vaccination and sera were prepared. MaxiSorb plates (NalgeneNunc International) were coated with Gallus gallus ovalbumine protein.After blocking with 1×PBS containing 0.05% TWEEN-20 and 1% BSA theplates were incubated with diluted mouse serum. Subsequently abiotin-coupled secondary antibody (Anti-mouse-IgG2a Pharmingen) wasadded. After washing, the plate was incubated with Horseradishperoxidase-streptavidin and subsequently the conversion of the ABTSsubstrate (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) wasmeasured.

11. Detection of an Antigen Specific Cellular Immune Response byELISPOT:

days after the last vaccination mice were sacrificed, the spleens wereremoved and the splenocytes were isolated. For detection of INFgamma acoat multiscreen plate (Millipore) was incubated overnight with coatingbuffer (0.1 M Carbonat-Bicarbonat Buffer pH 9.6, 10.59 g/l Na₂CO₃, 8.4g/l NaHCO₃) comprising antibody against INF□ (BD Pharmingen, Heidelberg,Germany). The next day 1×10⁶ cells/well were added and re-stimulatedwith 1 μg/well of relevant peptide (SIINFEKL of ovalbumine; SEQ ID NO;535); irrelevant peptide (Connexin=control peptide) or buffer withoutpeptide. Afterwards the cells are incubated for 24h at 37° C. The nextday the plates were washed 3 times with PBS, once with water and oncewith PBS/0.05% TWEEN-20 and afterwards incubated with a biotin-coupledsecondary antibody for 11-24h at 4° C. Then the plates were washed withPBS/0.05% TWEEN-20 and incubated for 2 h at room temperature withalkaline phosphatase coupled to streptavidin in blocking buffer. Afterwashing with PBS/0.05% TWEEN-20 the substrate (5-Bromo-4-Cloro-3-IndolylPhosphate/Nitro Blue Tetrazolium Liquid Substrate System from SigmaAldrich, Taufkirchen, Germany) was added to the plate and the conversionof the substrate could be detected visually. The reaction was thenstopped by washing the plates with water. The dried plates were thenread out by an ELISPOT plate reader. For visualization of the spotlevels the numbers were corrected by background subtraction.

12. Tumour Challenge:

One week after the last vaccination 1×10⁶ E.G7-OVA cells (tumour cellswhich stably express ovalbumine) were implanted subcutaneously in thevaccinated mice. Tumour growth was monitored by measuring the tumoursize in 3 dimensions using a calliper.

1. A polymeric carrier cargo complex, comprising: a) as a carrier apolymeric carrier formed by disulfide-crosslinked cationic components,and as a cargo at least one nucleic acid molecule.
 2. The polymericcarrier cargo complex according to claim 1, wherein the at least onenucleic acid molecule is an RNA.
 3. The polymeric carrier cargo complexaccording to claim 2, wherein the at least on nucleic acid molecule isan immunostimulatory nucleic acid.
 4. The polymeric carrier cargocomplex according to claim 2, wherein the at least on nucleic acidmolecule is an immunostimulatory RNA (isRNA) or an mRNA.
 5. A polymericcarrier cargo complex according to claim 2, wherein thenitrogen/phosphate (N/P) ratio of the cationic components to the atleast one nucleic acid molecule is in the range of 0.1-20, or in therange of 0.1-5, or in the range of 0.1-1.
 6. A polymeric carrier cargocomplex according to claim 1, wherein the polymeric carrier furthercomprises functional peptides or proteins.
 7. A polymeric carrier cargocomplex according to claim 6, wherein the functional peptides orproteins are peptide or protein antigens or antigen epitopes.
 8. Apolymeric carrier cargo complex according to claim 2, wherein thepolymeric carrier further comprises a ligand.
 9. A polymeric carriercargo complex according to claim 8, wherein the ligand is mannose. 10.The polymeric carrier cargo complex according to claim 2, wherein thecationic components are cationic peptides.
 11. The polymeric carriercargo complex according to claim 10, wherein the cationic peptides areselected from peptides according to formula (I)(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), whereinl+m+n+o+x=3-100, and l, m, n or o=independently of each other is anynumber selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90and 91-100, provided that the overall content of Arg, Lys, His and Ornrepresents at least 10% of all amino acids of the cationic peptide; andXaa is any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x=any numberselected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, and91-100 provided that the overall content of Xaa does not exceed 90% ofall amino acids of the cationic peptide, or are selected from peptidesaccording to subformula (Ia){(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa′)_(x)(Cys)_(y)} or frompeptides according to subformula (Ib)Cys₁{(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)}Cys₂ wherein(Arg)_(l); (Lys)_(m); (His)_(n); (Orn)_(o); and x are as defined above;Xaa′ is any amino acid selected from native or non-native amino acidsexcept of Arg, Lys, His, Orn; or Cys and y is any number selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90, and 91-100 providedthat the overall content of Arg (Arginine), Lys (Lys₁ ne), His(Histidine) and Orn (Ornithine) represents at least 10% of all aminoacids of the oligopeptide and wherein Cys₁ and Cys₂ are Cysteinesproximal to, or terminal to(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x).
 12. The polymericcarrier cargo complex according to claim 1, wherein the disulfide-bondsare formed by cysteine residues contained in the cationic peptides. 13.A polymeric carrier cargo complex according to claim 12, wherein thecysteine residue is located at the terminal ends of the cationicpeptides.
 14. A method of stimulating an immune response in a subjectcomprising administering to the subject a polymeric carrier cargocomplex according to claim
 1. 15. A vaccine comprising a polymericcarrier cargo complex as defined according to claim 1 and an antigen orRNA encoding an antigen.